棉花组织培养中异常苗的发生与转化

研究了棉花 (Gossypium hirsutum L.)组织培养中异常苗发生的类型、规律、影响因素及转化为正常苗的途径及技术。结果表明 ,在棉花组织培养中异常苗发生的频率极高 ,且形式多样 ,大体可分为生长点异常苗、叶片异常苗、胚轴异常苗、联体苗、丛生叶苗、单叶苗、玻璃化苗、白化苗、褐化苗和重新愈伤化苗等 1 0类。其中生长点异常苗和叶片异常苗最为常见。影响异常苗形成的因素很多 ,其中主要有外植体种类、培养基组成、培养方式和培养时间。有的异常苗来源于畸形胚 ,有的异常苗由正常苗转化而来。在合适的条件下异常苗可转化为正常苗 ,但不同培养基对异常苗转化为正常苗的影响不同 ,且不同基因型的异常苗转化为正常苗的能力也有差别。此外 ,从外界调控机制与棉花培养细胞的内在发育机制的相互作用方面讨论了造成棉花组织培养异常苗发生的原因 ;并讨论了降低异常苗产生频率的技术途径和异常苗存在对棉花组织培养的影响     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

文心兰试管苗分化及育苗技术研究

文心兰原球茎分化苗过渡驯化培养试验表明,改良1号+椰子汁100g/L的培养基有利于原球茎分化成苗,能促进分化苗生长健壮、整齐度好。壮苗与生根培养试验表明,以改良1号为基本培养基,加入香蕉泥100g/L、活性炭1g/L,在较强光照(2 000~3 000 lx)下培养,植株各项生长指标表现较好,有利于后期炼苗成活;以三角瓶为定瓶容器培养,植株接种效率最高、污染率最低;选取3cm高的植株定瓶,可显著提高文心兰工厂化组培育苗的工作效率。     

Slow desiccation leads to high-frequency shoot recovery from transformed somatic embryos of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L. cv. Coker 310 FR)

In Agrobacterium-mediated genetic transformation of cotton (Gossypium hirsutum L. cv. Coker 310FR) the frequency at which somatic embryos were converted to plantlets was significantly improved by subjecting the embryos to slow physical desiccation. We used Agrobacterium strain GV3101 containing the binary vector pGSFR with the nos-nptII gene for in vitro selection and the 35S gus-int fragment as a reporter to optimize the transformation protocol. Although the concentration of kanamycin was reduced during embryogenesis and embryo maturation, even at the lower levels somatic embryos were predominantly abnormal, showing hypertrophy and reduced or fused cotyledons or poor radicle ends. A majority of these embryos (more than 75%) were beta-glucuronidase (GUS)-positive. Embryos with an abnormal appearance showed a very poor conversion to plantlets. However, these embryos, when subjected to slow physical desiccation followed by transfer to fresh medium, regenerated single or multiple shoots from the cotyledonary end. These shoots could be grafted on wild-type seedling stocks in vitro, which, following their transfer to soil, developed normally and set seeds. Regenerated plants tested positive for the transgene by Southern analysis. An overall scheme for the high-frequency production of cotton transgenics from both normal and abnormal appearing somatic embryos is presented.     

Nodal segments or microtubers as explants for in vitro microtuber production of potato

Experiments on recycling small microtubers back into tissue culture revealed that they have a great advantage over nodal segments when used as explants in vitro. They produced plantlets ready for micropropagation in one-half the time it took nodal segments to do so. They did not require a fixed daylength (either long or short days) for this, nor were they dependent on the presence of sucrose in the culture medium. Small microtubers were more suitable than large because the latter produced very many branches which senesced more rapidly. When maintained in culture, these plantlets from microtubers themselves produced microtubers of a similar array of sizes and fresh weights to nodal explants, but at a much faster rate. For this, the presence of a high level of sucrose (8%) was beneficial, and slightly larger microtubers produced a higher yield. The microtubers produced from these plantlets were identical to those from nodal segments, and had a similar period af dormancy.     

樟叶越桔组培苗生根和移栽技术研究

为解决樟叶越桔(Vaccinium dunalianum)组培苗生根质量不佳、移栽成活率低的问题,该研究以樟叶越桔继代苗为材料,采用单因子试验从激素类型及浓度、培养基类型和蔗糖质量浓度对其生根的适宜条件进行筛选,进一步研究了不同基质配比对樟叶越桔移栽苗存活率的影响。结果表明:激素类型和浓度、培养基类型对樟叶越桔生根率的影响最大,其次为蔗糖质量浓度;最适合樟叶越桔生根的激素及浓度为IBA2.0 mg·L~(-1)、基本培养基类型为1/4MS、蔗糖质量浓度为15 g·L~(-1),樟叶越桔组培苗最佳生根培养基为1/4MS+IBA 2.0 mg·L~(-1)+活性炭0.1 g·L~(-1)+蔗糖15 g·L~(-1),生根率达100%,平均生根数为每株7.67条;根系呈辐射状、基部无愈伤组织,组培苗生长健壮、叶色浓绿;樟叶越桔组培苗移栽时以全腐殖土基质为佳,成活率为83.7%,植株叶片舒展,生长状况良好。该研究建立的优化体系有效地提高了樟叶越桔组培生根苗的生根率和生根质量,解决了后期移栽成活困难的问题,为优良的樟叶越桔植株规模化生产提供了科学依据和技术支持。     

Plantlet Regeneration from Protoplasts lsolated from an Embryogenic Suspension Cultureof Cotton(Gossypium hirsutum L.)

Protoplasts were isolated from an embryogenic suspension culture of commercial cotton cv. The protoplasts were released enzymatically and isolated by centrifugation on a sucrose cushion. The isolated protoplasts were initially cultured in a liquid medium with K3 mineral salts and modified Km8p organic compositions, supplemented with 0.05–0.1 mg/l 2,4-D, 0.2–0.5 mg/l 2ip in the dark. The regenerated plantlets from protoplasts of coker312 and coker 201 cv. were obtained. Embryogenesis from protoplast of Jin4 cv. and microcolonies form protoplasts of JiHe321 and Lul cv. were observed.     

葡萄试管苗热处理结合茎尖培养脱病毒效果的研究

对７个生食葡萄品种的试管苗热处理结合茎尖剥离培养脱除病毒的方法做了研究,结果表明,从热处理试管苗剥离的茎尖培养成活率为６１．２％,其中有１８．１％的成苗。各品种热处理试管苗剥离的茎尖分化再生能力不同：先形成愈伤组织的成苗率较低,而先分化芽的茎尖成苗率较高。这一方法对扇叶病毒和卷叶病毒的脱除率达９２％以上。     

澳洲青苹组织培养再生体系的研究

通过对澳洲青苹茎尖培养及植株再生体系中无菌培养物的建立、初代培养、继代培养、生根培养和移栽五个技术环节的研究,建立了澳洲青苹组织培养快速繁殖技术体系。结果表明：(1)BA0．3～0．6mg／L NAA0．1～0．3mg／L的MS培养基能促进澳洲青苹茎尖生长或大量侧芽的分化,不定芽生长健壮;(2)无根幼苗在含有IBA0．6～1．0mg／L NAA0．1mg／L的1／2MS培养基上暗培养一周后,再进行自然光培养,30d生根率可达到78%以上;(3)生根苗经50mg／L NAA浸渍1～2h后,移入蛭石：腐殖土：田园土=2：1：1的移栽基质中,保温保湿,成活率达80％以上;(4)进一步建立了自然光下生根、练苗一次完成的同步化体系,缩短了繁育时间,简化了步骤,降低了成本。此技术体系为澳洲青苹优良种苗快速繁殖提供了一条新途径。     

某些因素对玻璃苗形成的影响和玻璃苗在形态解剖上的特点

瑞香试管苗的玻璃化受到细胞分裂素浓度和茎尖外植体划、的影响,细胞分裂素浓度越高,茎尖外植体越小,产生的玻璃苗越多,玻璃苗与正常试管苗的茎尖在形态结构上有明显差别,主要表现为顶端分生组织原体原套结构异常和细胞的明显液泡化。     

组织培养中畸形胚状体及超度含水态苗的研究

畸形胚状和超度含水态苗是组织培养中常见的2种形态、生理异常现象,由于其难以发育成苗或很难移栽成活而严重影响组织培养的成功率。概述了这2种畸形现象的的形态特征及防止方法等,并讨论了应进一步研究的问题。     

Response of <Emphasis Type="Italic">Dionaea muscipula</Emphasis> J. Ellis to light stress in in vitro: physiological study

Hyperhydricity can cause significant economic loss for the micro-propagation industry that produces blueberry. In order to predict and control the occurrence of hyperhydricity, better understanding of the anatomical and physiological features of hyperhydric plantlets is required. In this study, we investigated the ultrastructural and physiological changes associated with hyperhydric blueberry plantlets. Compared to normal plantlets, hyperhydric plantlets exhibited reduced cell wall thickness, damaged membrane and guard cell structure, decreased number of mitochondria and starch granule, higher cell vacuolation, more intercellular spaces, and collapse of vascular tissues. In addition, excessive accumulation of reactive oxygen species (ROS) and ethylene, decreased stomatal aperture and water loss, as well as abnormity of stomatal movement were also evident in the hyperhydric plantlets. The results suggested that excessive ethylene and ROS produced in response to the stress arising from in vitro culture could lead to abnormal stomatal closure, causing the accumulation of water in the tissues. This would lead to subsequent induction of oxidative stress (due to hypoxia) and cell damage, especially guard cell structure, eventually giving rise to the symptoms of hyperhydricity. Reducing the content of ethylene and ROS, and protecting the structure and function of the stomata could be considered as potential strategies for inhibiting hyperhydricity or restoring the hyperhydric plants to their normal state.     

Observation of Androgenesis in Cultured Wheat Anthers at Meiosis,Tetrad, Early Uninucleate and Trinucleate Stage

The obtaining of calluses and plantlets from cultured wheat anthersat the stages from pollen mother cell to trinucleate microspore has been reported previously. Haploids as well as diploids existed among the regenerated plantlets derivedfrom anthers at these stages. Present paper reports the study on androgenesis patter-ns of cultured anthers at meiosis, tetrad, early mid- and late uninucleate and trinucleate stage. Cytological evidence of pollen-origin of calluses produced by anthers atthese stages was given. Observation showed that meiosis of wheat anthers was able tocomplete under culture conditions, resulting in releasing microspores, from which multinucleate and multicellular pollen grains formed. In meiosis anthers, abnormal cells,including syncytium and two kinds of binueleate calls were sometimes observed. Theymight be products of abnormal meiosis and abnormal development of tapetum cells. Itwas noted that failure and/or uncomplction of forming callus wall and/or pollen wallin in vitro anthers at meiosis, tetrad and early uninucleate stage occured often. Itmight lead to the low frequency of callus induction. Mature wheat anthers (trinucleate stage) contained both normal and abnormal pollen grains (pollen dimorphism); onlythe abnormal pollen grains developed into embryoids while all the normai trinucleatepollen grains degenerated rapidly. However, the date of the frequency of equal divisionof microspores suggested that abnormal pollen (N pollen, small pollen) could not be theonly source of androgenic pollens in cultured anthers at late uninucleate and other earlier stages.     

Regeneration of the barley zygote in ovule culture

An ovule culture technique has been established for barley that allows the regeneration of plants from zygotes. An average of 1.3 plantlets per ovule could be regenerated from more than 60% of the cultured ovules and about 75% of the regenerated plantlets developed into normal, fertile plants. The same regeneration frequencies were obtained in intact ovules and in ovules where the two integuments had been removed from the micropylar region. Unfertilized ovules and ovules where the fertilized eggs had been destroyed by a microinjection needle did not give rise to embryo-like structures. Plants could be regenerated from the zygote at the same frequency at developmental stages from immediately after fertilization until the formation of bicellular embryos. This tissue culture system appeared to be largely independent of genotype since similar regeneration frequencies were obtained in two different barley cultivars, Igri and Alexis, that in anther and microspore culture behave differently. The same technique has also been applied successfully in the wheat cultivar Walter.     

槐树试管苗在移栽驯化过程中叶表面结构的扫描电镜观察

用扫描电子显微镜观察了槐树试管试管苗在移栽驯化及大田生长过程中叶表面结构的变化。结果表明：随着移栽驯化及大田生长过程的延长,表皮细胞周缘突起增多,细胞之间相互嵌合,连结紧密;表皮蜡质结晶密度、长度及蜡质厚度逐渐增加,其结晶由“星”状转化为针状及棒状;气孔器密度及气孔开度由大到小,气孔器下陷程度增加。显示出试管苗在移栽驯化及田间生长过程中,叶表面结构对环境的适应性,其主要变化向着防止水分过度散失的方     

Meristem culture of Ophiopogon japonicus and production of embryo-like structures

Meristems of Ophiopogon japonicus (Liliaceae) were grown on a modified MS medium without auxins or cytokinins and plantlets and embryogenic callus were obtained at a low frequency. When meristems growing on modified basal medium were briefly soaked in 0.54 mM naphtaleneacetic acid (NAA) or temporarily grown on medium that contained NAA or NAA and benzyladenine, a larger proportion of meristems developed into plantlets or produced callus and additional plantlets following their return to basal medium. Calluses grown in liquid culture without auxins or cytokinins produced abundant single cells and cell aggregates. Larger cell aggregates formed embryo-like structures that produced roots, cotyledons, and then plantlets following transfer to soilid medium. Prolonged liquid culture produced embryo-like structures directly in liquid medium. These structures met many of the criteria for somatic embryos and developed into normal plantlets when placed on solid medium.     

Plant regeneration in Spanish cedar, <Emphasis Type="Italic">Cedrela odorata</Emphasis> L., using zygotic embryo explants from mature seed and improvement of embryogenic nodule initiation by heat shock

Conditions for induction of embryogenic nodules and subsequent somatic embryogenesis in the tropical hardwood Cedrela odorata are described. Embryo explants from ungerminated mature seeds were placed on Driver and Kuniyuki Walnut (DKW) medium using 0.8% w/v agar as the gelling agent, plus benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Various phytohormone combinations were tested, from which 5 μM BA + 50 μM 2,4-D were chosen as the standard regime based on a maximum frequency of embryogenic nodule occurrence of 20–25% on this medium. Nodules, when excised from the cotyledons and placed on growth-regulator-free medium, produced both plantlets and secondary embryogenic tissue. With extended culture on growth-regulator-free DKW medium, plantlets developed roots and could be transplanted into pots for further growth. The frequency of nodule initiation could be improved by either orienting the cotyledon explants with their lower (abaxial) surface in contact with the medium or imposing a pre-excision period of heat shock. The treatments together were additive. An optimum heat-shock temperature (47°C) and range of exposure times (8–12 h) were defined.     

江苏省棉区棉田杂草群落发生分布规律的数量分析

在对江苏省四大主产棉区８２个样点７２７块田地共５７．５ｈｍ＾２棉田杂草群落及草害进行了７级目测法调查采集数据后,对其进行主成分分析,并赋以生态学意义的解释,研究表明,江苏省棉田杂草的发生和分布与轮作种植制度和区域性化作制度导致的田间水分的巨大差异是决定杂草群落结构特征的最深刻的原因,导致全省水旱轮作棉 杂草群落有趋同性。     

江苏省棉区棉田杂草群落发生分布规律的数量分析

在对江苏省四大主产棉区８２个样点７２７块田地共５７．５ｈｍ＾２棉田杂草群落及草害进行７级目测法调查采集数据后,对其进行主成分分析（ＰＣＡ）,并赋以生态学意义的解释。研究表明,江苏省棉田杂草的发生和分布与轮作种植制度和地理区域性密切相关,其中轮作制度导致的田间水分的巨大差异是决定杂草群落结构特征的最深刻的原因,导致全省水旱轮作棉田的杂草群落有趋向性。而地理区域构成的土壤、气候等生态因子的显著影响表现     

植物幼苗玻璃化发生机制研究进展

玻璃化是指植物生长过程中出现的类似水浸状的半透明化生理病变现象,常见于植物组织培养。玻璃化现象是限制组培技术发展和组培苗商业化生产的三大主要影响因素之一,但是玻璃化的发生机制至今尚不清楚。以组培材料为对象研究玻璃化机制受多种人为因素的干扰,而以非组织培养材料为研究对象,避免了人为因素的干扰,有助于从本质上揭示玻璃化发生的分子机制。该文综述了非组培苗因软木脂含量改变、表皮蜡质合成减少、细胞膜过氧化受损、离子或水分子跨膜运输受阻等导致植物玻璃化的机制,以期为最终揭示植物玻璃化机制提供参考。