Thidiazuron对苜蓿愈伤组织的乙烯生成及其体细胞胚胎发生的影响

苜蓿叶柄在Ｂ５Ｈ培养基上诱导并继代所形成的是无色、松软的胚性愈伤组织、内含许多有分生能力的细胞团,当Ｂ５Ｈ中的ＫＴ用ＴＤＺ替换时,愈伤组织变绿变硬,出现管状分子和芽原基的分化,其体胚发生能力失,而且其乙烯生成和ＰＡＬ活性升高。乙烯形成酶的抑制剂ＣｏＣｌ２可以抑制ＴＤＺ的上述作用,部分地恢复愈伤组织的体胚发生能力,ＴＤＺ对愈伤组织的这些作用可能与其乙烯生成有关。     

提高小麦愈伤组织分化频率的因素

研究了影响小麦愈伤组织诱导、芽分化及其植株再生的一些因素。结果表明：在愈伤组织诱导和继代过程中添加ＡＢＡ（１．０ｍｇ／Ｌ）有利于小麦中晚期幼胚致密愈伤组织的诱导及再生能力的保持;外植体来源尤其是基因型对长期培养的愈伤组织再生能力有很大影响;不同的外源激素（ＫＴ、６－ＢＡ、ＩＡＡ、ＴＤＺ和玉米素等）也影响芽分化频率,其中ＴＤＺ可明显提高芽分化频率;在转入分化培养前对愈伤组织进行干燥处理可有效地提高其     

提高小麦愈伤组织分化频率的因素

研究了影响小麦愈伤组织诱导、芽分化及其植株再生的一些因素。结果表明：在愈伤组织诱导和继代过程中添加ＡＢＡ（１ ．０ｍｇ／Ｌ） 有利于小麦中晚期幼胚致密愈伤组织的诱导及再生能力的保持;外植体来源尤其是基因型对长期培养的愈伤组织再生能力有很大影响; 不同的外源激素（ ＫＴ、６ＢＡ、ＩＡＡ、ＴＤＺ和玉米素等） 也影响芽分化频率,其中ＴＤＺ可明显提高芽分化频率;在转入分化培养前对愈伤组织进行干燥处理可有效地提高其芽分化频率;在生根培养基中添加适量的ＩＡＡ 或ＮＡＡ 可有效促进生根。     

草莓离体叶片脱分化与再分化过程中的生理生化变化(简报)

在１．０ｍｇ．Ｌ＾－１ＴＤＺ与１．０ｍｇ．Ｌ＾－１．ＩＡＡ（培养基Ａ）作用下,草莓离体叶组织呼吸速率、蛋白质、核酸和糖类含量以及过氧化氢酸酥和苯丙氨酸解氨酶活性迅速提高,随即很快下降水平,愈伤组织不分化,在２．５ｍｇ．Ｌ＾－１ＴＤＺ与０．１ｍｇ．Ｌ＾－１ＩＡＡ（培养基Ｂ）作用下,上述指标行工提高,并维持在同一水平直至愈伤组织形成,其后大幅度提高,在愈伤组织分化芽前或分化时达到最大值。     

MGBG对苜蓿愈伤组织生长、体细胞胚胎诱导及其乙烯生物合成的影响

在苜蓿（ＭｅｄｉｃａｇｏｓａｔｉｖａＬ．）愈伤组织继代之初加入不同浓度的甲基乙二醛双（脒腙）（ＭＧＢＧ）（０．５,１,２ｍｍｏｌ／Ｌ）,可不同程度地抑制愈伤组织及其后体胚诱导悬浮培养物的生长和体胚形成;若在体胚诱导阶段加入,则上述的抑制作用更为强烈。ＭＧＢＧ不但促进愈伤组织乙烯释放量、内源ＡＣＣ（１氨基环丙烷１羧酸）水平和ＡＣＣ合成酶活性,也促进体胚诱导悬浮培养物的ＡＣＣ水平及ＡＣＣ合成酶的活性,但降低体胚诱导悬浮培养物的ＭＡＣＣ（丙二酰ＡＣＣ）水平。ＭＧＢＧ对体胚诱导和培养物的生长的抑制作用与其上述的对ＡＣＣ和乙烯生成促进作用及降低ＡＣＣ转化成ＭＡＣＣ的效应有关。     

荷兰芹胚性愈伤组织诱导及胚状体发生与内源ＩＡＡ和ＡＢＡ关系的初步研究

分析了荷兰芹胚性愈伤组织发生的条件,对Ｃo^2 作用下胚状体形成与培养物内源ＩＡＡ和ＡＢＡ的关系做了研究,结果表明,荷兰芹下胚轴胚性发生能力随其相对位置而异,自下而上逐渐提高,提高２,４－Ｄ浓度有利于胚性愈伤组织的诱导,水解酪蛋白对胚性能力的表达没有显影响。在胚状体发生过程中,添加Ｃo^2 显降低培养物内源ＩＡＡ和ＡＢＡ水平,并提高胚状体诱导率。其中对照组ＩＡＡ有一个峰值,ＡＢＡ有两个峰值;实验组ＡＢＡ水平,并提高胚状体诱导率。其中对照组ＩＡＡ有一个峰值,ＡＢＡ有两个峰值;实验组ＡＢＡ变化趋势与对照组相似,而ＩＡＡ始终凤有峰值出现,Ｃo^2 对ＩＡＡ和ＡＢＡ的抑制机制可能有所不同。     

天仙子(Hyoscyamus niger)花粉的胚胎发生与器官发生

天仙子花粉在附加不同浓度NOA的BN培养基中,形态发生能力不同,低浓度易由花粉直接形成胚状体,高浓度一般先形成愈伤组织,再由愈伤组织分化胚状体或根芽。实验表明,直接起源于花粉的胚状体与经花粉愈伤组织形成的胚状体,其胚胎发生的模式和顺序相同,均由原胚、球形胚、心形胚、鱼雷期胚到子叶期胚,但二者原胚的起源不同。花粉愈伤组织亦可经器官发生途径产生根芽。     

外源激素对金钱松胚外植体愈伤组织的诱导及其器官发生的调节作用

金钱松胚外植体在培养过程中由于外源激素的种类和配比的不同而存在着几种发育途径：直接从胚外植体表面分化不定芽;先诱导愈伤组织,再从愈伤组织分化不定芽;还可由愈伤组织分化出胚状体。激素ＢＡ对外植体不定芽的诱导起着关键作用。激素２,４－Ｄ则诱导愈伤组织,ＢＡ与２,４－Ｄ配比恰当诱导的愈伤组织分化出体细胞胚状体。　ＬＰ’附加低浓度的ＢＡ或ＫＴ（＜０．５ｍｇ／Ｌ）促进不定芽茎的伸长;　ＬＰ’附加浓度的ＩＢＡ（＜０．５ｍｇ／Ｌ）诱导不定根的发生。愈伤组织在基本培养基浓度为　×ＬＰ’或１×ＬＰ’的分化培养基上不定芽诱导率相似。     

MGBG对苜蓿愈作组织生长,体细胞胚胎诱导及其乙烯生物合成的影响

在苜蓿（ＭｅｄｉｃａｇｏｓａｔｉｖａＬ．）愈伤组织继代之初加入不同浓度的甲基乙二醛－双（脒腙）（ＭＧＢＧ）（０．５,１,２ｍｍｏｌ／Ｌ）可不同程度地抑制愈伤组织及其后体胚胎诱导悬浮培养物的生长和体胚形成,若在体胚诱导阶段加入,同上述的抑制作用更为强烈,ＭＧＢＧ不但促进愈伤组织乙烯释放量,内源ＡＣＣ（１－氨基环丙烷－１羧酸）水平和ＡＣＣ合成酶活性,也促进体胚诱导悬浮培养物的ＡＣＣ水平及ＡＣＣ合成酶的     

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。     

Genetic variability of somatic embryogenesis in tissue cultures of sugar beet breeding lines

Genetic variability of callus initiation and plant regeneration has been investigated among three sugar beet genotypes. It was found that TDZ has a genotype-independent effect on callus initiation and is responsible for more than a two-fold increase in the friable callus induction rate and more than a three-fold increase in the shoot regeneration rate from this callus. Along with the genotype-independent organogenesis, regeneration from callus occasionally went through the process of somatic embryogenesis in a highly genotype-specific manner. Despite fast and uncontrollable conversion of embryos to normal plants, it was possible to select and maintain repetitive embryogenic culture without loosing regeneration and root formation capabilities. Extensive experimenting with medium composition and culture conditions resulted in an optimal medium for maintenance of repetitive embryos. Comparing with BAP, low concentrations of TDZ provide higher level of adventitious shoot formation and do not induce vitrification of tissues.     

籼稻体细胞胚胎发生及器官发生的比较研究

在以幼穗为外植体的诱导培养及其以后的继代培养与悬浮培养中,从以下几个方面对３个籼稻品种的体细胞胚胎发生（Ｅ）与器官发生（Ｏ）的特点作了比较研究．在形态上,（Ｅ）型愈伤由胚状体组成,具有层次分明的结构．胚状体由胚芽、胚根及盾片组成．而（Ｏ）型的愈伤没有上述组织的分化．没有层次结构。植株再生能力方面．与器官发生途径相比较,体细胞胚胎发生途径之分化频率高．保持再生能力持续的时间长．从每个愈伤上发生的再生植株数多．（Ｏ）型愈伤结构较疏松．建立结构良好的、细胞团较小的悬浮细胞系容易．但这种细胞系在液体条件下几乎丧失再生能力．而（Ｅ）型愈伤则相反．建立好的悬浮细胞系．需要时间较长．但在悬浮条件下仍然具有再生植株的能力。在诱导条件方面．２,４－Ｄ及激动素分别作用时可诱导器官发生,共同作用时则诱导体细胞胚胎发生．再生植株途径方面,由于水稻同时具有器官发生及体细胞胚胎发生的特点．无论在诱导条件上还是在（Ｅ）与（Ｏ）本身的特点上均不似萝卜或烟草那样专一．并且有（Ｅ）与（Ｏ）相互转化和混合发生的复杂情况．     

In vitro morphogenic response in cotyledon explants of Semecarpus anacardium L.

Three different morphogenic responses??caulogenesis, direct somatic embryogenesis, and callusing??were noted in cotyledon explants of Semecarpus anacardium L. cultured in woody plant medium (WPM) containing thidiazuron (TDZ). Thidiazuron, at all concentrations tested, induced organogenic as well as embryogenic responses. The organogenic buds differentiated to shoots and the embryogenic mass (EM) gave rise to globular embryos which differentiated up to cotyledon-stage embryos on repeated culture in growth regulator (GR)-free WPM medium containing 0.2% activated charcoal after the removal of TDZ. The organogenic and embryogenic responses were optimal in 9.08???M TDZ after the removal of TDZ. Elongated shoots rooted in half-strength liquid WPM medium with 2.46???M indole butyric acid. Plants were successfully acclimatized and transferred to soil. Histological studies confirmed the direct origin of the organogenic buds from the cotyledon explants. The EMs produced somatic embryos on repeated culture in charcoal incorporated GR-free medium. Morphogenic callus formation from the cotyledon explants was also noted. This callus on repeated culture in WPM medium with charcoal differentiated into somatic embryos. Repetitive somatic embryogenesis was evident from direct and indirectly formed primary embryos. The somatic embryos did not convert into plantlets, though sporadic germination of embryos was observed through the emergence of roots.     

青扦胚性细胞悬浮培养中影响体细胞胚发生因素的研究

试验以青扦（Ｐｉｃｅａｗｉｌｓｏｎｉ）的胚性愈伤组织为材料,以改良５９基本成分附加２４－Ｄ１ｍｇ／Ｌ及ＫＴ１ｍｇ／Ｌ为培养介质,比较了液体悬浮与半固体二种培养方式对胚性愈伤组织增殖和体细胞发生的影响,研究了液体悬浮培养过程中影响体细胞胚发生的因素。结果表明：液体悬浮培养好于半固体培养,它的胚性愈伤组织的生长率为２６８％,是半固体培养的１２４倍;体细胞胚的分化率为９３％,是半固体培养的２２倍;悬浮培养较佳的培养条件为：初始细胞密度为２％（鲜重）,蔗糖浓度为２０ｇ／Ｌ,摇床转速为１００ｒ／ｍｉｎ,ｐＨ为５８。经过两个月悬浮培养,将培养物转至１／２改良５９附加ＡＢＡ１ｍｇ／Ｌ的分化培养基上,３个月后每ｇ培养物上可获得２８５个正常的子叶期体细胞胚。     

Cloning and expression of 1-aminocyclopropane-1-carboxylate oxidase cDNA induced by thidiazuron during somatic embryogenesis of alfalfa (Medicago sativa)

Embryogenic callus (EC) induced from petioles of alfalfa (Medicago sativa L. cv. Jinnan) on B5h medium turned green, compact and non-embryogenic when the kinetin (KN) in the medium was replaced partially or completely by thidiazuron (TDZ). The application of CoCl₂, which is an inhibitor of 1-aminocyclopropane-1-carboxylate oxidase (ACO), counteracted the effect of TDZ. Ethylene has been shown to be involved in the modulation of TDZ-induced morphogenesis responses. However, very little is known about the genes involved in ethylene formation during somatic embryogenesis (SE). To investigate whether ethylene mediated by ACO is involved in the effect of TDZ on inhibition of embryogenic competence of the alfalfa callus. In this study we cloned full-length ACO cDNA from the alfalfa callus, named MsACO, and observed changes in this gene expression during callus formation and induction of SE under treatment with TDZ or TDZ plus CoCl₂. RNA blot analysis showed that during the EC subcultural period, the expression level of MsACO in EC was significantly increased on the 2nd day, rose to the highest level on the 8th day and remained at this high level until the 21st day. However, the ACO expression in the TDZ (0.93 μM)-treated callus was higher than in the EC especially on the 8th day. Moreover the ACO expression level increased with increasing TDZ concentration during the subcultural/maintenance period of the callus. It is worth noting that comparing the treatment with TDZ alone, the treatment with 0.93 μM TDZ plus 50 μM CoCl₂ reduced both of the ACO gene expressions and ACO activity in the treated callus. These results indicate that the effect of TDZ could be counteracted by CoCl₂ either on the ACO gene expression level or ACO activity. Thus, a TDZ inhibitory effect on embryogenic competence of alfalfa callus could be mediated by ACO gene expression.     

Thidiazuron-induced organogenesis and somatic embryogenesis in sugar beet (Beta vulgaris L.)

Summary Improved in vitro tissue culture systems are needed to facilitate the application of recombinant DNA technology to the improvement of sugar beet germplasm. The effects of N ⁶-benzyladenine (BA) and thidiazuron (TDZ) pretreatment on adventitious shoot and somatic embryogenesis regeneration were evaluated in a range of sugar beet breeding lines and commercial varieties. Petiole explants showed higher frequencies of direct adventitious shoot formation and produced more shoots per explant than leaf lamina explants. TDZ was more effective than BA for the promotion of shoot formation. The optimal TDZ concentrations were 2.3–4.6 μM for the induction of adventitious shoot regeneration. Direct somatic embryogenesis from intact seedlings could be induced by either BA or TDZ. TDZ-induced somatic embryogenesis occurred on the lower surface of cotyledons at concentrations of 0.5–2μM and was less genotype-dependent than with Ba. A high frequency of callus induction could be obtained from seedlings and leaf explants, but only a few of the calluses derived from leaf explants could regenerate to plants via indirect somatic embryogenesis. These results demonstrated that TDZ could prove to be a more effective cytokinin for in vitro culture of sugar beet than BA. Rapid and efficient regeneration of plants using TDZ may provide a route for the production of transgenic sugar beet following Agrobacterium-mediated transformation.     

Endogenous ethylene in indirect somatic embryogenesis of <Emphasis Type="Italic">Medicago sativa</Emphasis> L.

Ethylene biosynthesis during different phases of somatic embryogenesis in Medicago sativa L. cv. Rangelander using two regeneration protocols, RPI and RPII, was studied. The highest ethylene production was detected during callus growth on induction medium in both regeneration protocols. Significantly less ethylene was produced by embryogenic suspension than by callus (RPII). Developing embryos synthesized higher amounts of ethylene than mature embryos. Production of ethylene was strongly limited by the availability of 1-aminocyclopropane-1-carboxylic acid and also by ACC-oxidase activity. However, removal of ethylene from culture vessels’ atmosphere using KMnO₄ or HgClO₄ had no significant effect on callus growth, somatic embryo induction and development. Reducing of ethylene biosynthesis by aminoethoxyvinylglycine substantially decreased somatic embryo production and adversely affected their development, indicating ethylene requirement during proliferation and differentiation but not induction.     

Wound-induced Accumulation of Trypsin Inhibitor Activities in Plant Leaves: Survey of Several Plant Genera

Asexual embryogenesis in Daucus carota L. `Queen Anne's Lace' callus was suppressed by Ethephon, ethylene, and 2,4-dichlorophenoxyacetic acid (2,4-D). The Ethephon effect could be attributed to volatile and nonvolatile substances. The volatile component was probably entirely ethylene. Ethylene was liberated in the cultures in direct proportion to Ethephon added to the medium. Autoclaving of Ethephon caused a substantial decrease of measurable ethylene. Continuous exposure of callus to 5 μl/l ethylene depressed somatic cell embryogenesis, but not markedly. Depression of embryogenesis by 2,4-D was unrelated to ethylene evolution.     

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree,Dalbergia sissoo Roxb

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.     

In vitro regeneration in Trifolium. 1. Direct somatic embryogenesis in T. rubens (L.)

The origin and development of zygotic and somatic embryos of Trifolium rubens L. was studied with the aid of paraffin sections and light microscopy. Zygotic embryos were collected, fixed and prepared daily from one to ten days after cross-pollination. Somatic embryos were obtained by plating petiole sections on modified L2 medium with 0.015 mgl^-1 picloram and 0.1 mgl^-1 6-BAP. Cultured petioles were collected and fixed daily from one to 25 days after plating. Two regions in the vascular bundle sheath of cultured petioles gave rise to callus. The first region was adjacent to the phloem fibers and produced friable callus. The second region gave rise to compact callus that was connected to the fascicular cambium. Somatic embryos originated from single cells in the cortex directly without intervening callus formation and from single cells in the friable callus. In addition, embryos arose from meristematic regions in compact callus. Many early stages of embryogenesis (one, two and four-celled stages) were observed in the cortex and friable callus. Zygotic embryogenesis in Trifolium differs from other legumes in that the suspensor is short and has a broad attachment. This arrangement was observed in zygotic embryos of T. rubens and in many somatic embryos. However, a continuum of somatic embryogenesis was observed where some young embryos had a Trifolium suspensor-like arrangement while others were attached to a long narrow suspensor-like structure more characteristic of Medicago.