Cell Ultrastructure of Kiwifruit (Actinidia chinensis) Shoot Tips During Cryopreservation

The changes in the cell ultrastructure of in vitro cultured shoot tips from dwarf genotype of kiwifruit （Actinidia chinensis Ganmi 5） during cryopreservation were investigated. Shoot tips were preserved in liquid nitrogen using vitrification, and the cell ultrastructure was examined using transmission electron microscopy （TEM）. The regular ultrastructure of the cell wall, cell membrane and nucleus of shoot tips could be damaged during the freezing and thawing associated with preservation using liquid nitrogen. The cell plasmolysis was increased and freezing tolerance was improved after precultufing and dehydrating in a preservation and vitrification solution （PVS2） （30% glycerol （Gly）＋ 15% ethylene glycol （EG）＋ 15% dimethylsulfoxide （DMSO） ＋ 0.4 mol L^-1 sucrose）. The structure of some cells with low degree of injury and reversible damage was similar to that of the control and they could undergo normal cell division and differentiation. Besides, they could recover automatically and regenerate after their reculture.

Response of carbohydrate metabolism-mediated sink strength to auxin in shoot tips of apple plants

Auxin(indole-3-acetic acid, IAA) has a considerable impact on the regulation of plant carbohydrate levels and growth, but the mechanism by which it regulates sugar levels in plants has received little attention. In this study, we found that exogenous IAA altered fructose(Fru), glucose(Glc), and sucrose(Suc) concentrations in shoot tips mainly by regulating MdSUSY1, MdFRK2, MdHxK1 and MdSDH2 transcript levels. Additionally, we used 5-year-old ’Royal Gala’ apple trees to further verify that these genes play primary roles in regulating sink strength. The results showed that MdSUSY1, MdFRK2, MdHxK1/3 and MdSDH2 might be major contributors to sink strength regulation. Taken together, these results provide new insight into the regulation of the carbohydrate metabolism mechanism, which will be helpful for regulating sink strength and yield.

Effect of Cytokinins on the Micropropagation of Carob(Ceratonia siliqua L.)through Shoot Tip Culture

In order to determine the most suitable cytokinin for the micropropagation of carob (Ceratonia siliqua L.), the effect of four cytokinins: BAP, zeatin, kinetin and 2-iP, was tested on explants derived from young seedlings of seven days. Organogenesis is better in the presence of BAP (0.5 mg/l), while buds growth is favored by zeatin (0.5 mg/l). The combination of the most suitable cytokinin (BAP at 0.5 mg/l) with IBA improves the caulogenesis when the concentration of this latter is low (0.1 mg/l);high concentrations of IBA have an inhibitory effect on elongation and neoformation of shoots and leaves. The multiplication and growth of shoots are more favorable on WPM medium in the presence of BAP (0.5 and 1 mg/l) alone or combined with 0.5 mg/l with GA3, while rooting is mainly favored by IBA, especially at 2 mg/l.

Influence of Ethylene Inhibitor Silver Nitrate on Direct Shoot Regeneration from in Vitro Raised Shoot Tip Explants of Sphaeranthus indicus Linn.—An Important Antijaundice Medicinal Plant

In the present investigation, an attempt has been made to study the influence of ethylene inhibitor silver nitrate on direct shoot regeneration in Sphaeranthus indicus, an important antijaundice medicinal plant, by using in vitro raised shoot tip explants. The effect of various concentrations of kinetin, BAP (0.5 - 3.0 mg/l), and NAA (0.1 - 0.5 mg/l) along with AgNO3 (0.1 - 1.0 mg/l) was studied. Among the combinations tested MS medium augmented with kinetin (1.0 mg/l), NAA (0.1 mg/l) and AgNO3 (0.4 mg/l) was found to be optimum for production of multiple shoots (34.3 ± 0.36). Addition of AgNO3 to the media not only increases shoot number in all the concentrations tested but also shoot length. AgNO3 at the concentration of 0.4 mg/l produced 35% more number of multiple shoots when compared to multiple shoots (10.8 ± 0.12) produced in control. In the present study by the addition of ethylene inhibitor silver nitrate and growth regulators, more number of multiple shoots (three folds) and shoot length was observed compared to control. These in vitro raised shoots were transferred to the rooting medium containing different concentrations of auxins such as NAA and IAA along with AgNO3 (0.1 - 0.6 mg/l). Better rooting response (21.6) was observed on NAA (2.0 mg/l) and AgNO3 (0.4 mg/l) containing media. The healthy rooted plantlets were transferred to polybags containing soil and vermiculate in 1:1 ratio for hardening. Finally the hardened plants were transferred to field environment for utmost survivability.

Effect of 24-Epibrassinolide on Growth of <i>in Vitro</i>Shoot Tips of Different Yam (<i>Dioscorea</i>Spp.) Species

In this work we compared the effect of the growth regulator content of the culture medium on the growth of in vitro shoot tips of five yam accessions belonging to four yam species (one Dioscorea alata, one D. rotundata, one D. cayenensis and two D. trifida). Medium S contained 0.6 μM benzyl adenine, 1.07 μM naphthalene acetic acid and 0.23 μM gibberellic acid while medium EBR contained 0.23 μM gibberellic acid and 0.1 μM 24-epibrassinolide. After 2 months of culture, oxidation level was significantly reduced on medium EBR compared to medium S for four of the five accessions tested. By contrast, medium EBR did not have any positive effect on shoot length since length of shoots produced after 2 months of culture on medium S and EBR were similar, except with accession 3-45T, for which shoot length was shorter on medium S compared to medium EBR. These results underline the potential of 24-epibrassinolide to reduce oxidation phenomena during in vitro culture and call for its utilization for regeneration of cryopreserved yam shoot tips, which is often impeded by oxidation phenomena.

“粉玉1号”草莓茎尖组织培养体系及其脱毒效果研究

“粉玉1号”是通过杂交育种方式选育的早熟抗病粉果草莓新品种。从大棚栽培的“粉玉1号”草莓植株上取匍匐茎芽为试料,通过外植体消毒、茎尖剥取、不定芽诱导和生根培养建立了“粉玉1号”茎尖组织培养脱毒技术体系,对茎尖培养苗和大棚栽培植株进行了草莓皱缩病毒(SCV)、草莓斑驳病毒(SMoV)、草莓轻型黄边病毒(SMYEV)和草莓镶脉病毒(SVBV)PCR检测。结果表明,“粉玉1号”草莓外植体灭菌方法为75%乙醇处理30 s,再用0.1%升汞溶液处理10 min;匍匐茎芽一般需剥去1片嫩叶和7片幼叶才能剥出茎尖;茎尖初代培养基宜采用MS+0.5 mg/L 6-BA,继代培养基宜采用MS+0.1 mg/L 6-BA,生根培养基宜采用不加植物生长调节剂的1/2 MS。大棚栽培植株样品存在SVBV感染,通过茎尖组培脱除了该病毒,SCV、SMoV和SMYEV在所有样品中均未检出。建立的茎尖脱毒技术体系可为“粉玉1号”草莓工厂化育苗提供参考。

基于外植体生理复幼的麻栎茎段无菌离体培养

以离体休眠枝条复幼处理后的萌芽茎段为外植体,开展20年生成龄麻栎的离体再生影响因素研究。结果表明:初代培养,以1/2 MS+3.0 mg/L 6-BA+2.0 mg/L KT+0.2 mg/L IBA为优选培养基,不定芽诱导率可达69.70%;继代培养,以1/2 MS+2.0 mg/L 6-BA+2.0 mg/L KT+0.2 mg/L IBA为优选增殖培养基,芽增殖系数约为2.0,但茎尖死亡率高,影响不定芽的连续继代。在影响继代苗茎尖死亡率的各试验因素中,培养基种类、继代转接时间、培养基中氯化钙浓度、硼酸浓度对继代培养中不定芽茎尖死亡率均有显著影响。WPM培养基中,继代培养23 d,硼酸质量浓度为12.4 mg/L,茎尖死亡率最低(13.06%)。在改良WPM+0.3 mg/L IBA+0.2 mg/L NAA的培养基上,成龄麻栎继代苗生根率最高(19.91%)。本试验为麻栎树种改良及无性快繁奠定了基础。

Improving Effect of Salicylic Acid on the Multipurpose Tree <i>Ziziphus spina-christi</i>(L.) Willd Tissue Culture

Salicylic acid (SA) is one of numerous phenolic compounds found in plants with numerous important physiological events. Exogenously application of Salicylic acid (SA) is useful for the growth and development of plants. This investigation aimed to study the improving effect of salicylic acid on the tissue culture of Ziziphus spina-christi. To study its improving effect were used shoot-tip explants cultured on Murashige and Skoog (MS) solid medium supplemented with different concentrations and various combinations of auxins, cytokinins and Salicylic acid. Media without growth regulators enhanced the growth and elongation of shoot-tip explants not its proliferation. Lower concentrations of cytokinin were better for shoot-tip proliferation than higher ones. 6-Benzylaminopurine (BAP) was superior to kinetin (KN) in shoot-tip proliferation. The optimum culture conditions for shoot-tip proliferation were achieved on MS medium supplemented with 0.25 mg/l 6-Benzylaminopurine (BAP) and 25 mg/l Salicylic acid (SA). The optimum culture conditions for callus formation were obtained on MS medium supplemented with 3 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 25 mg/l Salicylic acid (SA), while the optimum culture conditions for somatic callus formation were obtained on MS medium supplemented with 2.0 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 10 mg/l Salicylic acid (SA). The optimum culture conditions for rooting of shoots were obtained on MS medium supplemented with 0.25 mg/l of Indole-3-butyric acid (IBA) and 10 mg/l Salicylic acid (SA). The highest percentage of survival plants was obtained in the soil mixture supplied with 10 mg/l Salicylic acid (SA). Salicylic acid (SA) showed positive effect and good response on callusing, shooting and rooting of Z. spina-christi. Cultures received small amounts of salicylic acid were better than those lack it.

山丹茎尖玻璃化超低温保存技术体系的建立

为建立山丹(Lilium pumilum DC.)茎尖玻璃化超低温保存技术程序,以山丹无菌苗茎尖为试验材料,采用单因素试验方法,对玻璃化超低温保存技术的关键因素预培养基蔗糖浓度、预培养时间、装载处理时间、PVS2溶液处理时间进行依次筛选。最佳条件为:将茎尖接种于含0.5 mol/L蔗糖的预培养基中预培养7 d,装载处理20 min,PVS2溶液处理80 min后,保存于液氮中即可;可获得70.91%的茎尖存活率和51.85%的再生率,为山丹种质资源提供了简单易行、高效的长期保存方法。

不同IAA和6-BA质量浓度组合对甘薯茎尖培养和表型变异的影响

以甘薯品种烟薯29号的茎尖为外植体,MS培养基为基本培养基,研究不同质量浓度IAA(0.1、0.5 mg/L)和6-BA(0.1、0.5、1.0 mg/L)组合下愈伤组织诱导、芽分化、植株再生和表型变异情况。结果表明,MS培养基中IAA质量浓度为0.5 mg/L时对烟薯29号愈伤组织的诱导效果最佳,且利于茎尖成活,在此质量浓度下,平均愈伤组织诱导率和茎尖成活率分别达到了92.00%和88.00%。芽诱导率和丛生芽率随着培养基中6-BA质量浓度的升高总体上增加,在IAA质量浓度为0.1 mg/L的处理中,平均芽诱导率和丛生芽率分别达到了64.27%和80.56%。MS培养基中IAA质量浓度为0.5 mg/L时有利于植株再生,平均植株再生率达到了39.33%,在此质量浓度下,植株再生率和成苗数均随着6-BA质量浓度的升高而增加,成苗周期随着6-BA质量浓度的升高而缩短。烟薯29号的再生植株中出现了1株无性系变异植株,命名为YM6-11,其叶脉色、花冠形状和杂交亲和性与烟薯29号相比发生了改变。综上,烟薯29号茎尖培养最佳培养基组成为MS+0.5 mg/L IAA+1.0 mg/L 6-BA,将获得的再生植株用于实际生产时,应进行表型和遗传学鉴定,及早发现变异植株,减少不必要的经济损失。

茎尖离体培养小黄姜变异株品质特征及转录组学分析

本研究采用恒温干燥法、水蒸气法和HPLC等方法分析茎尖离体培养小黄姜变异株的干物质含量、精油含量、姜辣素含量等品质特性,并利用高通量测序技术和生物信息学方法进行转录组学分析。结果表明,小黄姜突变株干物质、纤维素含量显著低于正常株,分别为13%和0.6%;木质素、精油及姜辣素含量分别为正常株的1.5倍、1.54倍和1.26倍。转录组学分析共获得43989条Unigene的注释结果,13342个SSR标记。获得DEG 338条,其中,上调表达164条,下调表达174条。有信息注释DEG的239条,分别在GO、COG、KEGG、KOG、Pfam、Swiss-Prot、eggNOG等功能数据库注释到DEG 132条、88条、81条、130条、186条、171条和224条。DEGs主要集中在光合作用碳的固定(GOT1)、植物激素信号传导(AXU/IAA、B-ARR、ERFL1/2、MYC2)、萜烯类和姜辣素等次生代谢物的合成(CHS)、植物生理节律(CRY)等生物途径。由此可知,AXU/IAA、B-ARR、ERFL1/2、MYC2等基因的差异表达导致小黄姜突变株的精油含量、姜辣素含量以及其他农艺性状发生改变,故在栽培前期可以通过对相关基因的表达差异分析确定植株属性,从而区分正常株与突变株。

草莓茎尖组培繁育体系优化及SRAP遗传稳定性检验

以组培苗遗传稳定和植株健壮为标准,优化草莓茎尖组培繁育体系,以期为草莓工厂化育苗提供科研和技术依据。以‘凤冠’和‘天仙醉’草莓为材料,设置植物生长调节剂浓度梯度,调整培养基基础配方,调查茎尖分化情况、增殖苗形态、增殖系数、生根苗叶片及根系等性状,并比对组培前后植株SRAP标记扩增条带的差异。筛选出初代培养基配方MS+6-BA 0.5 mg/L+NAA 0.01 mg/L,增殖培养基配方MS+6-BA 0.3 mg/L+NAA 0.01 mg/L,生根培养基配方1/2MS+硝酸钙0.45 g/L;组培前后植株DNA使用19对SRAP引物特异性扩增,得到的72个基因位点条带未发生变化,以上条带形成的DNA指纹没有差异。研究得到的草莓茎尖组培繁育体系具备植物生长调节剂种类少且浓度低、继代次数少、增殖系数低、组培时间短、组培苗健壮且基因稳定等特征。

包埋干燥超低温保存苹果离体茎尖

选用继代培养７０ｄ的苹果离体茎尖，在５℃条件下低温驯化３周，经不同浓度蔗糖预培养及藻酸钠包埋后，在无菌空气中干燥脱水４ｈ直接投入液氮，化冻后茎尖存活率达７０％以上。通过选择材料的生理状态可缩短和简化保存过程。

甜樱桃茎尖培养及PNRSV的RT-PCR检测

研究了茎尖大小、接种方式、培养基成分、试材基因型对甜樱桃品种茎尖培养的影响。 1年生成熟枝条上茎尖成苗率为 8.3%～ 2 3.7% ,嫩梢上的茎尖成苗率为 2 7.3%～ 37.5 %。利用RT PCR技术对部分甜樱桃试管苗进行了早期病毒鉴定 ,筛选出一些不带李坏死环斑病毒 (PNRSV)的甜樱桃试管苗 ,并证明甜樱桃试管苗微茎尖培养不能有效脱除PNRSV。

香蕉茎尖的玻璃化法超低温保存及其植株再生

以香蕉（Musa spp.）为试材,对其离体培养茎尖玻璃化法超低温保存影响因素进行研究.结果表明,不定芽在MS＋3.0～5.0 mg/L 6-BA＋0.1 mg/L NAA的培养基上分化较好.香蕉茎尖超低温保存较佳体系是：2.0～3.0 cm的茎尖在含0.4 mol/L蔗糖培养基上预培养2 d,剥取带1～2个叶原基的茎尖（长1.0～1.5 mm）,室温（25℃）下装载液（MS＋2 mol/L甘油＋0.4 mol/L蔗糖）装载20～30 min,然后用玻璃化溶液（PVS2）于0℃下处理40 min,换1次PVS2后迅速投入液氮.保存至少1 h后,在40℃水浴中化冻90 s,用1.2 mol/L蔗糖培养液洗涤2次,每次10 min,然后转入含0.3 mol/L蔗糖的MS培养基上,暗培养10～15 h后转移到含0.5 mg/L 6-BA的MS培养基中,暗培养1周后转移到正常光下,3个香蕉品种（巴西蕉、广东香蕉2号、广东粉蕉1号）的成活率分别为75.9%、40.0%和69.6%,再生率分别为63.4%、35.0%和63.4%.再生植株生长和分化正常,生根后可移栽成活.

草莓组培快繁及叶片诱导植株再生的研究

以4个草莓品种的匍匐茎尖为起始材料,试验以MS为基本培养基附加不同激素草莓茎尖诱导植株再生、增殖、生根的培养基,确立了草莓组培快繁技术方法;又以4个草莓品种的组培苗叶片为外植体,探讨了不同激素配比、基因型对诱导草莓不定芽再生的影响;通过进一步试验,以MS+B5有机为基本培养基附加6 BA2 253mg/L、IAA1 752mg/L,探讨草莓不同基因型及不同外植体对诱导不定芽再生的影响,初步建立了一个有效的、较高频率的芽再生系统,也为基因的转化找到一个较好的受体材料。

草莓试管内诱导匍匐茎和高温处理结合茎尖培养脱毒技术研究

【目的】获得可用于生产的无病毒草莓种苗。【方法】在试管内对深山草莓花瓣愈伤组织分化产生的优良变异新品种的试管苗进行匍匐茎诱导,并采取试管内高温处理结合茎尖培养对该品种进行脱毒研究,应用均匀设计法对各步骤主要影响因子进行优化筛选。【结果】该草莓新品种试管苗匍匐茎诱导的适宜培养基为:LS+6-BA1.00mg/L+GA31.90mg/L+KT1.00mg/L,其诱导率为99.5%以上;将含有匍匐茎的试管苗在试管内于56℃条件下培养85d后,切取1mm的茎尖在培养基MS+6-BA0.50mg/L+NAA0.20mg/L中进行茎尖愈伤组织诱导,然后将茎尖愈伤组织转接到培养基MS+6-BA1.00mg/L+NAA0.05mg/L中进行愈伤组织芽苗再分化培养,对再生芽苗进行病毒检测,脱毒率为100%。【结论】利用试管内高温处理结合茎尖脱毒的方法可以完全脱除草莓病毒,从而建立了草莓脱毒体系。

文心兰茎尖组织培养的研究

文心兰茎尖离体培养研究表明 :文心兰茎尖在 MS+6 - BA 3mg/ L +Ad2 .5～ 3.5 mg/ L +NAA 0 .2 mg/ L培养基上培养 4 5 d后 ,茎尖分化出芽的同时也形成较多的原球茎 ;原球茎在 MS+6 - BA 2 .0 mg/ L +Ad 0 .5 mg/ L +NAA 0 .1mg/ L培养基上增殖速度最快 ,生物量增殖系数可达 8.8713;来自不同增殖培养基上增殖的原球茎在相同的分化培养基上培养时 ,接种后 15 d观察 ,不同来源的原球茎分化率不同 ,30 d后的分化率仍存在一定的差异 ;分化的文心兰幼苗在 1/ 2 MS+NAA 0 .2 mg/ L生根效果最好。

甜樱桃品种微繁体系的建立及优化

对影响甜樱桃微繁殖的多种因素进行了研究,建立起甜樱桃品种高效、稳定的微繁殖技术体系。从1年生成熟枝条上剥取1mm茎尖在培养基上可以分化成苗。去顶芽嫩梢的增殖系数显著高于未去顶芽嫩梢的增殖系数。培养基中无机氮含量和NH4+/NO3-比例对于甜樱桃试管苗增殖继代具有重要影响,降低NH4+/NO3-比例有利于产生叶片形态正常的试管苗植株。两步生根法,即先在附加4.0mg/LIBA的1/2MS培养基上暗培养6d,然后转移到不附加激素的1/2MS培养基上继续培养,可以使顽童和早红宝石的生根率达到100%。试管苗移栽成活率达到86.5%。