大蒜愈伤组织诱导条件初报

以白皮大蒜和大头蒜为材料,报道了大蒜品系、外植体的低温前处理、外植体大小及取材种类不同对愈伤组织诱导的影响。结果表明：（１）无论是４℃低温保藏或室温保存前处理,大头蒜的消毒效果均优于白皮大蒜,４℃低温前处理可降低外植体出愈污染;（２）只有带表皮的外植体才可诱导愈伤组织,外植体较大（０．５ｃｍ×０．３ｃｍ×０．２ｃｍ）,继代期愈伤组织产生量较多;外植体偏小（０．２ｃｍ×０．１ｃｍ×０．１ｃｍ）,极少产生愈伤组织;（３）４℃冷藏１８ｄ对大头蒜愈伤组织诱导有一定影响,但对白皮大蒜影响不大。冷藏可推迟出愈１～２ｄ。     

甘蔗原生质体的植株再生

从甘蔗（Ｓａｃｃｈａｒｕｍ ｏｆｆｉｃｉｎａｒｕｍ Ｌ．）嫩叶外植体诱导愈伤组织,经继代培养后,挑选胚性愈伤组织,转入ＭＳ３ 液体培养基,进行悬浮培养。当培养物分离出小粒状的细胞团,细胞变得小而圆时,用于分离原生质体。原生质体以琼脂糖固化的培养方式培养于ＭＲＰ１ 培养基中。由原生质体再生的愈伤组织有两种类型。挑选粒状、坚实的再生愈伤组织转移到Ｎ６ 分化培养基上,“新台糖１ 号”再生的愈伤组织,在含有ＫＴ ０．５ ｍ ｇ／Ｌ的培养基中,分化出绿芽并长成完整的植株。而“粤糖５７－４２３”和“ＵＳ６６－５６－９”再生的愈伤组织,在加有０．１％ 的活性炭的培养基中,前者分化出白化苗,后者分化出根     

烯效唑对玉米幼胚培养的影响

以玉米幼胚为外植体,研究了烯效唑对玉米幼胚愈伤组织诱导以及愈伤组织分化成植株的影响。结果表明,烯效唑(S-3307)的浓度越高玉米幼胚胚芽的生长受抑制作用越强,胚性愈伤组织发生率越高,有的基因型幼胚对过高浓度的烯效唑表现出不适反应,以至于胚性愈伤转化成非胚性愈伤;不同基因型的玉米幼胚在不同浓度的烯效唑影响下愈伤组织诱导率差异较大;烯效唑的浓度越高胚性愈伤组织分化率也高,但不同基因型对烯效唑的反应不尽相同。     

影响农杆菌介导玉米愈伤组织遗传转化因素的研究

用农杆菌介导玉米愈伤组织的转化,其筛选的结果得到的抗性愈伤组织受玉米愈伤组织的继代时 间、浸染的农杆菌菌液浓度、共培养的温度以及其共培养时间等因素的影响。玉米愈伤继代后7～9d,农杆菌 浓度为OD600值0.3左右、共培养温度约22℃、培养时间3d时,抗性愈伤的获得率最高。     

抗赖氨酸加苏氨酸莲体细胞变异体的筛选

对籽莲红花建莲（Ｎｅｌｕｍｂｏｎｕｃｉｆｅｒａｃｖ．Ｈｏｎｇｈｕａｊｉａｎｌｉａｎ）和白心湘莲（Ｎ．ｎｕｃｉｆｅｒａｃｖ．Ｂａｉｘｉｎｘｉａｎｇｌｉａｎ）杂交而成的幼胚的子叶、胚轴及幼胚叶（芽）形成的愈伤组织,施以赖氨酸加苏氨酸胁迫培养３０天,从中筛选出抗性愈伤组织并形成再生植株。低浓度的赖氨酸加苏氨酸促进愈伤组织的生长,而高浓度的赖氨酸加苏氨酸则抑制愈伤组织生长,直至具有致死作用,这种致死作用是因为高浓度的赖氨酸加苏氨酸抑制了天冬氨酸合成途径中的天冬氨酸激酶和高丝氨酸脱氢酶造成的,细胞发生变异后对赖氨酸和苏氨酸产生了抗性,即与天冬氨酸合成途径有关的氨基酸增加。抗性愈伤组织与未胁迫的愈伤组织的氨基酸测定表明,在总计１７种氨基酸中,抗性愈伤组织有１４种氨基酸含量超过原始愈伤组织,１种持平,２种不及原始愈伤组织。再生抗性植株的莲籽氨基酸测定显示,在１７种氨基酸中,有１２种超过母本红花建莲,１５种超过父本白心湘莲。     

农杆菌介导的玉米自交系愈伤组织转化条件的优化

采用农杆菌介导法,将外源基因导入玉米骨干自交系掖515和掖502胚性愈伤组织并再生玉米植株。在农杆菌浸染玉米胚性愈伤组织过程中,采取真空渗入、部分酶解和超声波处理,可以显著提高转化率。农杆菌浸染玉米胚性愈伤组织时,在50kPa下真空渗入5min,掖515和掖502胚性愈伤组织的转化率分别从6.3％和4.5％提高到8．7％和7．8％。愈伤组织在农杆菌浸染前,以0．2％离析酶酶解10min,掖515和掖502胚性愈伤组织的转化率分别提高到8.3％和8．9％。在农杆菌浸染时,以功率100W超声波处理90-120s,掖515和掖502胚性愈伤组织的转化率最高可达9．1％和9．4％。     

玉米愈伤组织对草丁膦的抗性及氨基酸对这种抗性的影响

将玉米（ＺｅａｍａｙｓＬ．）（Ｐ９１０×Ｚ３１）愈伤组织接种于含不同浓度草丁膦及不同氨基酸的Ｎ６培养基上,测定愈伤组织重量增长及ＮＨ＋４积累水平。统计结果表明,当培养基中草丁膦浓度为４ｍｇ／Ｌ时,愈伤组织重量增长最低;浓度为８ｍｇ／Ｌ时,ＮＨ＋４积累水平达最高。综合考虑这两种因素,在培养基上选择转化体的草丁膦浓度以６ｍｇ／Ｌ为宜。在含有草丁膦的选择培养基上,Ｌ精氨酸和Ｌ谷氨酸能通过代谢调节显著降低玉米愈伤组织中的ＮＨ＋４浓度;而Ｌ脯氨酸虽明显促进玉米愈伤组织在选择培养基上的重量增长,但并不降低玉米愈伤组织中的ＮＨ＋４浓度     

软枣猕猴桃试管苗叶片和茎段的愈伤组织诱导及植株再生

从软枣猕猴桃试管苗茎段和叶片诱导出愈伤组织并得到再生植株。茎段外植体容易愈伤化,但其愈伤组织难以分化,叶片外植体不易愈伤化,但其愈伤组织容易分化。ＭＳ培养基分别附加ＢＡＰ、Ｋｉｎ、ＴＤＺ或ＣＰＰＵ都不能诱导芽的分化,而ＭＳ附加玉米素能有效地诱导芽分化,其中以２．０ｍｇ／Ｌ玉米素效果最好。     

不同基因型玉米愈伤组织诱导与植株再生研究

以5个玉米品系幼胚为外植体,研究了基因型、2,4-D浓度以及胚龄对愈伤组织诱导的影响;6-BA对愈伤组织分化的影响;以及IBA对再生芽生根的影响。结果表明：除。31外,其他基因型的外植体在相同条件下均可诱导出愈伤组织,但是不同基因型间存在显著差异;2,4-D浓度和胚龄显著影响愈伤组织的诱导,且2,4-D浓度为2．0mg／L,胚龄在11—13d之间时,玉米愈伤组织诱导率较高且质量较好。将愈伤组织转入分化培养基后,6-BA促进了愈伤组织的再分化;在生根培养基中,IBA促进了再生芽生根,经过炼苗后移栽获得再生植株。     

鹤望兰组织培养与工厂化快繁程序的研究

将材料接种于诱导愈伤组织手芽的培养基上,培养２个月后,胚芽外植体下出现白色颗粒状的愈伤组织,４个月后愈伤组织上出现小芽丛。将小芽丛转入不加植物激素的ＭＳ培养基上,芽的生长加快,２个月左右可长成３－６ｃｍ高的丛小植株。将小植株切下,插入根培养基中,一般３５ｄ左右基部突出很小的白色根尖。     

In vitro Reaction of Sunflower (Helianthus annuus L.) to the Toxin(s) Produced by Alternaria alternata, the Casual Agent of Brown Leaf Spot

A bioassay system was developed for studying the in vitro reaction of sunflower ( Helianthus annuus L. cv. 'Nanus') against the toxin produced by the virulent pathotype IMI 366417 (1) of the pathogenic fungus Alternaria alternata. Cotyledons from 2-week-old seedlings were cultured on a MS (Murashige and Skoog) medium supplemented with 0.3 μM NAA (α-napthylacetic acid) and 1.3 μM BA (6-benzyladenine). Exponentially growing calli were transferred to selective media containing toxin solutions at various concentrations. The fresh weight of the cultured calli was reduced as the toxin concentration increased, although the viability of the cells, expressed as callus dehydrogenase activity, increased. Selection for toxinresistant genotypes was attempted at 30% toxin concentration, which causes a 90% reduction in callus growth. After one month in culture, 18% of the calli demonstrated resistance to the toxin. However, no plants could be regenerated from those calli after transfer onto a MS medium supplemented with 5.4 μM NAA and 4.4 μM BA. The effect of the toxin purification method on toxin yield and biological activity, as well as its possible mode of cellular action are discussed. The results of these experiments may contribute to a better understanding of the disease mechanism and help establish an efficient selection method of resistant sunflower genotypes.     

通过组织培养筛选小麦抗赤霉病突变体的研究

选用中抗赤霉病的春小麦品种和品系的花药进行离体培养,以小麦赤霉病29号菌株产生的致病培养滤液为选择剂,结合物理诱变处理,进行抗病鉴定,用赤霉病菌分生孢子直接接种在愈伤组织和再生植株筛选抗病突变体。在83块愈伤中有53块抗病。在11株的再生植株中有9株均比未经培养滤液处理的对照提高了抗病性,从中选出4株抗病性接近或超过“苏麦3号”品种。     

用PEG法把外源基因导入甘蓝型油菜原生质体再生转基因植株

通过PEG处理把外源基因导入甘蓝型油菜原生质体。转化介质中二价阳离子的种类和浓度、携带DNA及PEG溶液的pH值都会影响基因导入效率。以潮霉素抗性和卡那霉素抗性作标记,均成功地筛选到了抗性愈伤组织。相对转化频率分别为1.3%和0.2%。前者明显高于后者。把抗性愈伤组织转到分化培养基上,分化出芽。诱导生根后移栽到土壤中,生长状况良好。酶活性测定和Southern blotting分析表明外源基因已插入到植物细胞基因组中。该遗传转化系统存在的主要问题是抗性愈伤组织分化频率较低。本文对其原因作了初步分析。     

Valine-resistant plants from in vitro selected tobacco cells

Summary Growth inhibition of protoplasts and cells of tobacco (Nicotiana tabacum L.) occurs when 0.1 to 10 mM L-valine is present in the culture medium but not if L-isoleucine is simultaneously added. Mesophyll protoplasts from haploid tobacco were submitted to ultraviolet irradiation and cell colonies derived from these protoplasts were plated in a medium containing valine. Plants were regenerated from five presumptive resistant calli and retested for valine-resistance. Among the recovered plants two were definitely resistant to valine. This trait was transmitted in sexual crosses as a mendelian character.     

用基因枪法将人工雄性不育基因导入小麦的研究初报

利用ＰＤＳ１０００／氦气基因枪将人工构建的雄性不育基因（ＴＡ２９－Ｂａｒｎａｓｅ基因）导入小麦栽培品种豫责１８号的幼胚细胞。然后在含有１０～２０ｍｇ／Ｌ除草剂Ｂａｓｔａ的培养基础上筛选与分化。从１７０个幼胚中获得６株绿苗,对照的７０个幼胚中未得到绿苗。对其中３株已生根且长势好的绿苗进行Ｓｏｕｔｈｅｍ杂交分析,结果表明,这３株绿苗皆为转基因植株,转化效率达１．８％。     

Atrazine and diuron resistant plants from photoautotrophic protoplast-derived cultures of Nicotiana plumbaginifolia

Atrazine and diuron resistant clones were isolated from diploid photoautotrophic protoplastderived colonies of Nicotiana plumbaginifolia. Protoplasts were mutagenised with 0.1 mM N-ethyl-N-nitrosourea and colonies were screened for resistance after plating. Selection of calli was carried out on their ability to grow and green on a selective medium containing either atrazine or diuron. Plants were regenerated from most tolerant calli. Herbicide spray showed that plants of 6 and 4 clones were resistant to atrazine and diuron, respectively.Abbreviations Atrazine 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine - diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - NEU N-ethyl-N-nitrosourea - PSII photosystem II     

Selection of NaCI Tolerant Calli and Regeneration of Plants in Maize

The embryonic calli produced from immature embryos of inbred “Huangzhao-4” of maize, that had been maintained for half a year, were transferred to media supplemented with different NaC1 concentrations (5, 10, 15, 20, 25, 30g/L) for callus selection. NaCl tolerant calli were established through three generations of selections. The growth and frequency of survival calli were affected significantly by NaCl concentration. The proliferetion of NaCl-tolerant calli was relatively good on medium containing of 10g/L NaC1. From these calli, plant lets could be produced on differentiation medium. On medium supplemented with 10g/L of NaC1 the plantlets could normally grow to transplantation. In NaCl-tolerant calli cultured on medium containing 10g/L of NaC1, the contents of free amino acids, free proline, Na+, K+ were 18.0%,87.3%,661.9%,25.5% respectively higher than those in un-selected calli grown on subculture medium, but Ca2+ content decreased significantly. On medium containing 10g/L of NaC1, cells and their organelles in NaCl-tolerant calli had normal morphology and structure, and vigorous metabolism, but in un-selected calli, the majority of cells turned to wards dying. Although tolerant plants regenerated and their filial ones had grown in non-salted soil, their progenies retained the property tolerance, but showed segregation of the degrees of tolerance. In 10g/L NaC1 solution, the seeds of progenies from one plant regenerated could germinate normally, and grow into healthy seedlings. Therefore, the NaCl-tolerant calli and plantlets that we have obtained NaCl-tolerant variants.     

In vitro selection of barley and wheat for resistance against Helminthosporium sativum

Summary Calli derived from immature embryos of barley and wheat genotypes were screened for their resistance to purified culture filtrate produced by the fungus Helminthosporium sativum P.K. and B. Two selection methods were used: a continuous method in which four cycles of selection were performed one after another on toxic medium and a discontinuous method in which a pause on non-toxic medium was given after the second or third cycle of selection. The latter was superior as it allowed the calli to regain their regeneration ability. About 3,000 calli from two barley genotypes and 2,000 from two wheat genotypes were used for selection. The selection with the pathotoxins resulted in 6% to 17% surviving calli. Toxin tolerant callus lines of barley were characterised by protein isozymes. Zymograms showed one more isozyme than with the unselected sensitive callus. Barley and wheat plants have been regenerated from callus lines surviving the toxin treatment and in vivo testing against pathogen revealed that the majority of these plants were less sensitive.     

芦笋抗S—（2—氨乙基）—L—半胱氨酸（AEC）变异体的筛选

S-(2-氨乙基)-L-半胱氨酸(AEC)可抑制芦笋愈伤组织的生长,此抑制作用可被赖氨酸或甲硫氨酸部分解除。用0.5mmol/L的AEC进行筛选,得到抗性愈伤组织AR10并再生植株。AR10愈伤组织经一年多的继代培养,在离开选择剂组培继代两代后仍保持对AEC的抗性。抗性系愈伤组织还表现出对2mmol/L的半胱氨酸具交叉抗性,对1mmol/L的赖氨酸加苏氨酸表现部分交叉抗性。AR10再生植株一部分保持对AEC的抗性,而一部分则无抗性。对抗性愈伤组织及其再生植株的氨基酸分析表明,愈伤组织内游离赖氨酸、苏氨酸、甲硫氨酸都有增加,而在再生植株内却发现半胱氨酸和赖氨酸的特异性增加,分别是对照植株的5.4和4.6倍。