Apical meristem transcriptome analysis identifies a role for the blue light receptor gene GhFKF1 in cotton architecture development

Cotton architecture is determined by the differentiation fate transition of axillary meristem(AM),and influences cotton yield and the efficiency of mechanized harvesting.We observed that the initiation of flowering primordium was earlier in early-maturing than that in late-maturing cultivars during the differentiation and development of AM.The RNA-Seq and expression level analyses showed that genes FLAVIN BINDING,KELCH REPEAT,F-BOX1(GhFKF1),and GIGANTEA(GhGI)were in response to circadian rhythms,and involved in the regulation of cotton flowering.The gene structure,predicted protein structure,and motif content analyses showed that in Arabidopsis,cotton,rapseed,and soybean,proteins GhFKF1 and GhGI were functionally conserved and share evolutionary origins.Compared to the wild type,in GhFKF1 mutants that were created by the CRISPR/Cas9 system,the initiation of branch primordium was inhibited.Conversely,the knocking out of GhGI increased the number of AM differentiating into flower primordium,and there were much more lateral branch differentiation and development.Besides,we investigated that proteins GhFKF1 and GhGI can interact with each other.These results suggest that GhFKF1 and GhGI are key regulators of cotton architecture development,and may collaborate to regulate the differentiation fate transition of AM,ultimately influencing plant architecture.We describe a strategy for using the CRISPR/Cas9 system to increase cotton adaptation and productivity by optimizing plant architecture.

The AGL6-like gene OsMADS6 regulates floral organ and meristem identities in rice

Although AGAMOUS-LIKE6 (AGL6) MADS-box genes are ancient with wide distributions in gymnosperms and angiosperms, their functions remain poorly understood. Here, we show the biological role of the AGL6-1ike gene, OsMADS6, in specifying floral organ and meristem identities in rice (Oryza sativa L.). OsMADS6 was strongly ex- pressed in the floral meristem at early stages. Subsequently, OsMADS6 transcripts were mainly detectable in paleas, lodicules, carpels and the integument of ovule, as well as in the receptacle. Compared to wild type plants, osmads6 mutants displayed altered palea identity, extra glume-like or mosaic organs, abnormal carpel development and loss of floral meristem determinacy. Strikingly, mutation of a SEPALLATA (SEP)-like gene, OsMADS1 (LHS1), enhanced the defect of osmads6 flowers, and no inner floral organs or glume-like structures were observed in whorls 2 and 3 of osmadsl-z osmads6-1 flowers. Furthermore, the osmadsl-z osmads6-1 double mutants developed severely indetermi- nate floral meristems. Our finding, therefore, suggests that the ancient OsMADS6 gene is able to specify "floral state" by determining floral organ and meristem identities in monocot crop rice together with OsMADS1.

Determination of the Photoperiod-Sensitive Inductive Phase in Maize with Leaf Numbers and Morphologies of Stem Apical Meristem

It is vital to determine the effective photoperiods of maize for making full use of tropical germplasm, which is the foundation for determining the effect of latitude and planting date on the development of photoperiod-sensitive maize cultivars. The objective of this study is to determine the photoperiod-sensitive inductive phase using reciprocal transfer between long- day （LD） （15 h d^-1） and short-day conditions （SD） （9 h d^-1）. For Huangzao 4 and CML288, days to tassel and pollen shedding were recorded, and stem apical meristems （SAM） were observed by a laser scanning confocal microscope. The results show that the seedlings are insensitive to photoperiod when they are very young （juvenile）. However, after this period, LD delays flowering and increases the leaf numbers below the inflorescence, and the length of the interval of the photoperiod-sensitive inductive phase is longer under LD conditions than under SD conditions. Transferred from SD to LD, plants show a sudden decrease in leaf numbers once sufficient SD has been received for flower commitment. While transferred from LD to SD, plants have a continuous increase in leaf numbers during the photoperiod sensitive inductive phase under LD conditions. At the same time, when plants are competent to flowers, the obvious morphology is the elongation of maize SAM. There is an obvious variance of the photoperiod sensitive phase under LD and SD conditions in different maize.

AtCDC5 regulates the G2 to M transition of the cell cycle and is critical for the function of Arabidopsis shoot apical meristem

As a cell cycle regulator, the Myb-related CDC5 protein was reported to be essential for the G2 phase of the cell cycle in yeast and animals, but little is known about its function in plants. Here we report the functional characterization of the CDC5 gene in Arabidopsis thaliana. Arabidopsis CDC5 （AtCDC5） is mainly expressed in tissues with high cell division activity, and is expressed throughout the entire process of embryo formation. The AtCDC5 loss-of-function mutant is embryonic lethal. In order to investigate the function of AtCDC5 in vivo, we generated AtCDC5-RNAi plants in which the expression of AtCDC5 was reduced by RNA interference. We found that the G2 to M （G2/M） phase transition was affected in the AtCDC5-RNAi plants, and that endoreduplication was increased. Additionally, the maintenance of shoot apical meristem （SAM） function was disturbed in the AtCDC5-RNAi plants, in which both the WUSCHEL （WUS）- CLAVATA （CLV） and the SHOOT MERISTEMLESS （STM） pathways were impaired. In situ hybridization analysis showed that the expression of STMwas greatly reduced in the shoot apical cells of the AtCDC5-RNAi plants. Moreover, cyclinB1 or Histone4 was found to be expressed in some of these cells when the transcript of STM was undetectable. These results suggest that AtCDC5 is essential for the G2/M phase transition and may regulate the function of SAM by controlling the expression ofSTMand WUS.

The GhREV transcription factor regulate the development of shoot apical meristem in cotton(Gossypium hirsutum)

Background:Manual topping is a routine agronomic practice for balancing the vegetative and reproductive growth of cotton(Gossypium hirsutum)in China,but its cost-effectiveness has decreased over time.Therefore,there is an urgent need to replace manual topping with new approaches,such as biological topping.In this study,we examined the function of Gh REV transcription factors(a classⅢhomeodomain-leucine zipper family,HD-ZIPⅢ)in regulating the development of shoot apical meristem(SAM)in cotton with the purpose of providing candidate genes for biological topping of cotton in the future.Results:We cloned four orthologous genes of At REV in cotton,namely Gh REV1,Gh REV2,Gh REV3,and Gh REV4.All the Gh REVs expressed in roots,stem,leaves,and SAM.Compared with Gh REV1 and Gh REV3,the expression level of Gh REV2 and Gh REV4 was higher in the SAM.However,only Gh REV2 had transcriptional activity.Gh REV2 is localized in the nucleus;and silencing it via virus-induced gene silencing(VIGS)produced an abnormal SAM.Two key genes,Gh WUSA10 and Gh STM,which involved in regulating the development of plant SAM,showed about 50%reduction in their transcripts in VIGS-Gh REV2 plants.Conclusion:Gh REV2 positively regulates the development of cotton SAM by regulating Gh WUSA10 and Gh STM potentially.

The Wheat Plastochron Mutant, fushi-darake, Shows Transformation of Reproductive Spikelet Meristem into Vegetative Shoot Meristem

In wheat plants at the vegetative growth stage, the shoot apical meristem (SAM) produces leaf primordia. When reproductive growth is initiated, the SAM forms an inflorescence meristem (IM) that differentiates a series of spikelet meristem (SM) as the branch. The SM then produces a series of floret meristem (FM) as the branch. To identify the mechanisms that regulate formation of the reproductive meristems in wheat, we have investigated a leaf initiation mutant, fushi-darake (fdk) which was developed by ion beam mutagenesis. The morphological traits were compared in wild type (WT) and fdk mutant plants grown in the experimental field. WT plants initiated leaves from SAM at regular intervals in spiral phyllotaxy, while fdk plants had 1/2 alternate phyllotaxy with rapid leaf emergence. The fdk plants have increased numbers of nodes and leaves compared with WT plants. The time interval between successive leaf initiation events (plastochron) was measured in plants grown in a growth chamber. The fdk plants clearly show the rapid leaf emergence, indicating a shortened plastochron. Each tiller in fdk plants branches at the upper part of the culm. The fine structure of organ formation in meristems of fdk plants was examined by scanning electron microscopy (SEM). The SEM analysis indicated that fdk plants show transformation of spikelet meristems into vegetative shoot meristems. In conclusion, the fdk mutant has a heterochronic nature, i.e., both reproductive and vegetative programs were simultaneously in operation during the reproductive phase, resulting in a shortened plastochron and transformation of reproductive spikelets into vegetative shoots.

Floral Organogenesis and Ring Meristem in <i>Phytolacca</i>

To further study the floral organogenesis and discussing the floral origin of Phytolacca, the procedures of floral organogenesis were observed in Phytolacca esculenta and Phytolacca zhejiangensis. The results showed that the floral organogenesis was consistent in Phytolacca. Their sepals were 2/5 helix, and with counter-clockwise and clockwise, usually the first sepal located at non-median of abaxial side. The first sepal of Phytolacca esculenta was initiated at non-median of adaxial side. There was no evident relationship between sepal and stamen initiating position, and the stamens initiated on ring meristem, they initiated approximately at the same time, and when the androecium member was numerous, they initiated centrifugally, the outer stamen initiated irregularly. Carpel initiated alternately with inner stamens. And the carpels connected by septum, if the septum grew more, the carpel was syncarpous at morphology, otherwise the carpel was apocarpous at morphology. So the syncarpous and the apocarpous have no successively relationship on evolution. Ovule initiated inside the carpel and opposite to carpel. Androecium, carpel and ovule initiated at ring meristem.

Efficient Regeneration System for Genetic Transformation of Mulberry (<i>Morus indica</i>L. Cultivar S-36) Using <i>in Vitro</i>Derived Shoot Meristems

Shoot meristems used for the study were exercised from the in vitro regenerated shoots cultured on MS medium supplemented with 0.5 mg/L of BAP for multiplication. The sensitivity of the in vitro regenerated was studied using shoot meristems of 0.5 cm. Shoot meristems were cultured on medium containing 10-100 mg/l kanamycin to determine the concentration that was lethal for multiple shoot induction and root induction. The response of shoot multiplication decreased (66.2%-6.2%) as the concentration of kanamycin increased (10.0-70.0 mg/L) with complete inhibition of shoot proliferation at 100 mg/L kanamycin. The rooting phase was very sensitive to kanamycin compared to shoot multiplication. The percentage of shoots that rooted decreased (53.8%-4.8%) with increase in the concentration of kanamycin (10.0-70.0 mg/l) on IBA and 2,4-D supplemented medium. For transformation studies, the shoot tips that were infected with Agrobacterium strain were placed on selection medium containing MS medium with 0.5 mg/L BAP and 100 mg/L kanamycin and scored for the putative transformed shoots. An average of 62.2% of shoot tips developed shoot buds from the base and the shoots reached a length of 0.5-1.0 cm at the end of 30 days of culture on the selective medium in comparison to control which showed no response. An average of 66.7% of the regenerated plants showed GUS expression on selection medium where 43.2% and 65% of GUS expression was recorded in the leaves and callus. Leaves and callus induced from the controls did not show GUS activity. Stable integration of nptII gene with the genomic DNA from these transformed plants was confirmed through PCR analysis. Our result presents an efficient regeneration system using in vitro derived shoot meristems for Agrobacterium mediated gene transfer.

Generation of Virus Free Potato Plantlets through Meristem Culture and Their Field Evaluation

Different aspects of micropropagation through meristem culture for the production of virus indexed source plants, in vitro tuberization and field evaluation of the in vitro regenerated plants were studied on four commercial cultivars of potato (Solanum tuberosum L.) viz., Diamant, Cardinal, Shilbilati and Lalpakri. The investigation was conducted at Rajshahi, Bangladesh from December 2010 to March 2012 to produce virus-free potato plantlets through meristem culture, shoot multiplications with root induction as well as their acclimatization and evaluation of morphological characters and tuber yield under field condition. Shoot tips of 25 - 30 day old field-grown plants of above mentioned four cultivars were used for meristem isolation. After isolation, meristems of these varieties of potato were cultured on “M” shaped filter paper bridge in Murashige and Skoog (MS) liquid medium. Four different treatments of media formulations viz. 0.1 mg/L KIN + 0.1 mg/L GA3, 0.1 mg/L KIN + 0.5 mg/L GA3, 0.5 mg/L KIN + 0.1 mg/L GA3 and 0.5 mg/L KIN + 0.5 mg/L GA3 were used as plant growth regulators. From these formulations MS + 0.1 mg/L KIN + 0.5 mg/L GA3 was found to be the best for the primary establishment of meristem culture. The primarily established meristems were subcultured on to MS semisolid basal medium supplemented with four different treatment combinations of hormones viz. 0.5 mg/L BA + 1.0 mg/L IBA;0.1 mg/L KIN + 0.1 mg/L GA3;0.5 mg/L BA + 0.5 mg/L GA3 and 0.5 mg/L KIN + 0.5 mg/L GA3 were used to identify the suitable media compositions for shoot proliferation. Results showed that out of these four media treatments the formulation 0.5 mg/L BA + 0.5 mg/L GA3 was found to be the best suitable for shoot generation. Among the four cultivars of potato higher frequency of shoot proliferation (number of shoots/explant and longest shoot length) was observed in Diamant, though the highest shoot formation (76%) was recorded in Cardinal. Virus free in vitro grown potato plantlets were obtained through DAS-ELISA test and used substantially for micro-propagation. After gradual acclimatization of rooted plantlets of four potato cultivars, they were transferred into the field for cultivation and established successfully. It was observed from the field study of in vitro meristem-derived plantlets that there were no virus-affected plants. The virus-free exotic varieties were much superior in all vegetative attributes and yield compared to those of indigenous varieties with producing potato plants of normal height. In contrast, the indigenous varieties took a longer time to tuber initiation and maturity, lower plant height and number of leaves per plant, a higher number of tubers but a lower amount of tuber weight per plant, and poorer tuber grade than the exotic varieties. However, the variety Cardinal exposed the best performances in the context of survival percentage of plantlets (90%), days to tuber initiation (DTI), a number of leaves per plant (NL), tuber weight per plant (343.40%) and the percentage of rich tuber grade.

The Effects of Lead on the Meristem of Wheat Seedlings

The ultrastructure of apical meristem cells was studied in Triticum aestivum L. cv. “Trizo” seedlings grown on soil without or enriched with selenium and survived 14 days’ stress caused by lead pollution in the soil. The soil treatments: control—the original soil;(Pb1)—50 mg&middot;kg&minus;1;(Pb2)—100 mg&middot;kg&minus;1;(Pb1 + Se1) —0.4 mg&middot;kg&minus;1 Se added to Pb1 treated soil;(Pb1 + Se2)—0.8 mg&middot;kg&minus;1 Se added to Pb1 treated soil;(Pb2 + Se1)—0.4 mg&middot;kg&minus;1 Se added to Pb2 treated soil;(Pb2 + Se2)—0.8 mg&middot;kg&minus;1 Se added to Pb2 treated soil were used. Light and other conditions were optimal for plant growth. A distinctive feature of the cells of the apical meristem of control plants was the absence of nuclear membranes. Proplastids were membrane vesicles 1 - 2 microns in diameter, filled with contents of varying degrees of density, from membrane vesicles containing only plastid DNA up to a fully formed structure of proplastids. In (Pb1)-plants, the amount of cytoplasmic ribosomes and proplastids in the meristematic cells was less than in the control. The structure of the forming proplastids was almost the same as that of the control plants. Signs of degradation of meristematic proplastids, such as a decrease of their diameter, observed in (Pb2)-plants. The introduction of selenium into lead contaminated soil increased the accumulation of Pb in plants, especially in the roots and apical meristem. In (Pb1 + Se1)-, (Pb1 + Se2)-, (Pb2 + Se1)-, and (Pb2 + Se2)-plants, the number of cytoplasmic ribosomes in meristematic cells increased, which indirectly indicates an increase in protein synthesis. Based on our concept about the formation (assembly) of proplastids in the cells of the apical meristem, we believe that toxic agents, such as lead, which inhibit the development of proplastids into chloroplasts in mesophyll cells, act on apical meristem cells at the stage when plastid DNA is replicated in the cytoplasm and is not yet surrounded by a plastid membrane.

Barley FASCIATED EAR genes determine inflorescence meristem size and yield traits

In flowering plants,the inflorescence meristem(IM)provides founder cells to form successive floral meristems,which are precursors of fruits and seeds.The activity and developmental progression of IM are thus critical for yield production in seed crops.In some cereals,such as rice(Oryza sativa)and maize(Zea mays),the size of undifferentiated IM,which is located at the inflorescence apex,is positively associated with yield traits such as spikelet number.However,the relationship between IM size and yieldrelated spike traits remains unknown in the Triticeae tribe.Here we report that IM size has a negative correlation with yield traits in barley(Hordeum vulgare).Three FASCIATED EAR(FEA)orthologs,HvFEA2,HvFEA3,and HvFEA4,regulate IM size and spike morphogenesis and ultimately affect yield traits.Three HvFEAs genes are highly expressed in developing spikes,and all three loss-of-function mutants exhibit enlarged IM size,shortened spikes,and reduced spikelet number,which may lead to reduced grain yield.Natural variations identified in HvFEAs indicate selection events during barley domestication.We further reveal that HvFEA4,as a transcription factor,potentially targets multiple pathways during reproductive development,including transcriptional control,phytohormone signaling,and redox status.The roles of barley FEA genes in limiting IM size and promoting spikelet formation suggest the potential of increasing yield by manipulating IM activity.

B Class Floral Homeotic Genes are Involved in the Petal Identity and Flower Meristem Determinations in Chrysanthemum morifolium

Chrysanthemum morifolium,an ornamental crop with diverse forms of inflorescence,is a good model for studying flower development in Asteraceae.However,the genetic background is complex and the mechanisms of regulating flower development are still unclear.Here,we identified two natural mutant lines of chrysanthemum and named them M1 and M2 according to the severity of the phenotype.Both lines showed defects in petal identity,and the petals of the M1 line had a mild phenotype:partially loss of petal identity and conversion of petals into green,leaf-like organs.The M2 line had severe phenotypes:in addition to severe petal defects,secondary inflorescences were produced in the capitulum to replace the normal ray and disc florets,which indicated a transformation of a flower meristem into an inflorescence meristem.Transcriptome sequencing of WT and M2 inflorescences was performed and found altered expression of floral organ development A,B and E class genes,where B and E class genes were significantly down-regulated.qRT-PCR analysis in both M1 and M2 lines revealed that the expression of three chrysanthemum class B genes CmAP3.1,CmAP3.2 and CmPI,was negatively correlated with phenotypic severity.This suggests that class B genes in chrysanthemum not only have conserved functions in determining petal identity but also were involved in the determinacy of the flower meristem.This study provides insights into the functions of class B genes in flower development,and is informative for dissecting the molecular mechanisms of flower development in chrysanthemum.

Multi-effects and Mechanism of Broad Beau M_1 Root-tip Cells Implanted by Low Energy N^+ Beam

Broad beans were divided into six groups and implanted with N+ beam of 30 KeV, 8 × 1016/cm2 per time for various radiating times respectively. Besides the statistics of its vigor of germination, the M1 root-tip cells of these broad beans were systematically analyzed on their changes in mitotic percentage, morphology and behavior of chromosomes, along with the structure o f cytoskeletons, including microtubule and intermediate filament. Based on all results of these studies, our opinions have been expressed in the report on the mechanism of low-energy N+ beams effecting on higher dicotyledons such as broad beau.

激素调控植物分枝/分蘖的研究进展

分枝/分蘖是植物株型的一个重要特征,也是腋芽起始和芽生长的结果,对经济作物的种子产量以及牧草产量均具有决定性作用。多种激素及其互作效应在植物分枝/分蘖的发生和生长发育过程中起着关键的调控作用,且环境因素也是通过改变植物体内激素含量及其平衡调控分枝的。本研究综述了多种激素对植物分枝/分蘖调控机制的多个方面,包括生长素、细胞分裂素、独脚金内酯、油菜素内酯、脱落酸和赤霉素、乙烯、茉莉酸及不同激素信号相互作用形成的复杂调控网络,旨在为利用激素调控机制培育具有理想株型的高产新作物品种奠定基础。同时分析了激素机制调控植物分枝/分蘖的现存问题,并展望了激素调控植物分枝/分蘖的研究方向,以期为通过激素调控改良作物株型提供理论依据。

玉米的腋生分生组织及其发育模式调控

玉米的腋生分生组织在玉米生长过程中作为次要生长轴,部分腋生分生组织发育为雌穗,经过授粉后最终形成果穗。腋生分生组织的发育赋予了玉米极强的可塑性,直接决定了植株的形态结构和最终产量。本文从玉米腋生分生组织的形态建成、发育模式、活性维持和发育调控方面综述了研究进展。玉米腋生分生组织营养生长时期受TB1基因的抑制基本没有侧枝的发育,生殖生长时期形成雌穗,其发育属于“分离分生组织”模型,多个茎节的叶腋位置均有腋生分生组织形成,自上而下开始发育,当正常的发育模式被打破时会产生空秆或多穗现象,造成减产。腋生分生组织形成后主要依靠CLV-WUS负反馈通路等调控途径维持活性,生长素介导的顶端优势是腋生分生组织生长过程中主要内源调控因素,其合成、转运以及相关信号转导都决定了腋生分生组织的生长状态。此外,多种营养元素也通过参与不同的生化和生理过程影响腋生分生组织的发育。进一步揭示玉米腋生分生组织调控网络,明晰腋生分生组织间相互作用关系,优化玉米形态结构,对玉米常规和分子育种均具有重要理论意义。

向日葵YABBY基因家族鉴定及表达分析

【目的】鉴定向日葵中花发育相关的HaYABBY基因,有助于探究其在向日葵花发育过程中的生物学功能和调控机制,为向日葵品种选育提供重要的分子线索。【方法】基于向日葵基因组和转录组数据,使用生物信息学方法,分析向日葵YABBY基因家族成员的染色体定位、系统进化关系、基因结构、保守基序、蛋白结构及理化性质、启动子顺式作用元件和组织表达模式等,并通过RT-qPCR分析HaYABBY基因在WT向日葵和花分生组织决定和花器官分化缺陷突变体cb1中st2-st8时期的表达差异。【结果】在向日葵基因组中鉴定到12个YABBY基因家族成员,分属于4个亚家族,分布在8条染色体上,并且同一亚家族中成员在基因结构和保守基序组成上高度相似。其中8个基因HaYABBY1a、HaYABBY1c、HaYABBY3、HaYABBY4b、HaYABBY5a、HaYABBY5b、HaCRABS CLAWa、HaCRABS CLAWb在向日葵花发育时期特异性高表达。在这8个基因中,HaYABBY3、HaYABBY5a和HaYABBY5d在突变体cb1花发育后期表达上调,且与WT花发育早期水平相当;而HaYABBY4b和HaCRABS CLAWa在cb1中的表达被强烈抑制。顺式作用元件分析显示,HaYABBY均含有多个启动子顺式作用元件,部分元件仅存在于特定基因中。其中,胚乳表达元件存在于HaCRABS CLAWa和HaYABBY4a中;生长素响应元件存在于HaCRABS CLAWa、HaCRABS CLAWb、HaYABBY4a、HaYABBY4b和HaYABBY5c中;分生组织表达元件存在于HaYABBY1a、HaYABBY1c、HaYABBY4b、HaYABBY5a和HaYABBY5d中;赤霉素响应元件存在于HaCRABS CLAWa、HaYABBY5b、HaYABBY5c和HaYABBY5d中;种子特异性调控元件存在于HaYABBY5a、HaYABBY5c和HaYABBY5d中。【结论】HaYABBY3、HaYABBY4b、HaYABBY5a、HaYABBY5d和HaCRABS CLAWa等5个HaYABBY基因在花分生组织的转变和花器官发生过程中可能发挥重要作用,它们在花分生组织转变和花器官发生调控中可能受到生长素响应因子ARF、赤霉素响应因子MYB以及MADS-box等关键因子调节。

小肽调控植物分生组织发育的机制及其在作物改良中的研究进展

在高等植物的生长发育过程中,根尖分生组织和茎尖分生组织不断分裂分化产生根系、茎、叶和花等器官,分别形成植物的地上和地下部分。小肽是胞间通讯的关键信号分子之一,参与了植物生长发育、抗病抗逆等诸多生命活动,其中包括植物根尖和茎尖分生组织的干细胞活性调控,进而影响了植物器官的生成和发育。近年来,越来越多的研究揭示了小肽分子在分生组织维持和发育中的重要性,及其在多种作物农艺性状调控中的关键作用。本文综述了小肽及其受体在拟南芥和作物中维持分生组织稳态的分子机制,探讨了小肽对作物产量性状的影响及在作物改良中的应用策略,并对小肽领域的研究方向进行了分析和展望。

茶菊茎尖分生组织高效脱毒再生体系优化

【目的】筛选适宜热处理时间和脱毒剂,优化茶菊茎尖分生组织高效脱毒再生体系,解决脱毒苗获得率低的问题。【方法】以6个茶菊品种无菌苗为材料,采用昼/夜40℃/32℃变温、化学脱毒剂及其组合预处理无菌苗,比较茎尖分生组织的出愈率、再生频率和平均再生芽数及不同方法脱毒效果。【结果】茎尖分生组织再生频率在不同品种茶菊间差异显著,并以‘婺源皇菊’最高(54.77%);茎尖分生组织再生在不同昼夜变温热处理时长间差异显著,以‘杭白菊’、‘七月雪’再生效果最好。‘婺源皇菊’茎尖分生组织再生在脱毒剂水杨酸、利巴韦林、脱落酸处理下均受到抑制,在水杨酸、利巴韦林结合变温热处理下得到促进,在高浓度脱落酸结合热处理下受到严重抑制,且10μmol/L水杨酸结合热处理的促进效果最好。在适宜热处理、化学脱毒剂结合热处理可获得茶菊完全脱毒苗。【结论】10μmol/L水杨酸浓度结合40℃/32℃昼/夜变温预处理40d能显著提高茎尖分生组织再生频率,获得脱毒苗。

赤霉素对根尖径向生长的调节作用研究

通过转基因与RNAi基因沉默技术,获得了由35S组成型启动子和TobRB7根尖特异表达型启动子分别启动表达的PtGA20ox、PtGA2ox1烟草转基因株系和NtGA20oxRNAi株系,对转基因植株根尖进行大样本(n>100)显微观察。统计结果显示:转基因植株根尖径向生长与对照相比具有极显著差异,外源施用GA3与赤霉素合成抑制剂PAC可以逆转这种差异,表明赤霉素对根尖径向生长具有负调控作用;此外,利用TobRB7启动子在根尖分生组织特异上调和下调内源赤霉素水平可以得到与组成型调控转基因植株相似的表型,表明赤霉素对根尖径向生长的负调控作用主要是通过对根尖分生组织生长发育的调控实现的。

甘蓝型油菜根系突变体lrn1、prl1和野生型根系显微结构的差异

油菜外源细胞分裂素不敏感突变体lrn1和prl1表现为磷高效。营养液培养0.2μmol/L细胞分裂素(6-BA)处理,与甘蓝型油菜野生型‘宁油7号’(WT)相比,突变体lrn1侧根较多,prl1主根较长。本研究利用体式显微技术、非切片压片法以及石蜡切片等技术,对3个基因型在ddH2O和0.2μmol/L 6-BA处理下的根毛、根表皮细胞分化及根尖解剖结构的差异进行了观察,结果表明:ddH2O处理,种子发芽后第1、3、6、9 d,lrn1、prl1和WT根尖成熟区根毛较少。0.2μmol/L 6-BA处理,种子发芽后第3 d,lrn1、prl1和WT根尖形成大量根毛,其中WT根毛最多、密度最大;prl1根毛最少,密度也最小;lrn1处于两者之间。种子发芽后第6 d,lrn1、prl1和WT分生区和伸长区明显缩短,lrn1和prl1分生区面积无显著差异,但两者均显著大于WT;lrn1和prl1根冠细胞结构较正常,而WT根冠细胞结构畸形;lrn1皮层原细胞之间排列较WT和prl1紧密。种子发芽后第9 d,lrn1已有4条侧根,但prl1与WT无侧根形成。6-BA处理,prl1主根较长,与其根尖分生区面积较大密切相关;lrn1侧根较多,可能与中柱原细胞排列密度较高密切相关。