In vitro propagation using adventitious buds technique as a source of new variability in chrysanthemum

Eleven cultivars of Chrysanthemum × grandiflorum (Ramat.) Kitam.: ‘Richmond’ and its 10 radiomutants, representing the Lady group, were propagated in vitro with shoot tips and leaves as explants. The aim of this study was to investigate if the explant type used for micropropagation affects the genotype and phenotype of chrysanthemums. Plants grown from shoot tips and adventitious buds formed on leaves were rooted in vitro, acclimatized and cultivated in glasshouse up to full-flowering. The colour and shape of inflorescences of plants obtained from two different explant types were compared within the cultivars. All plants derived from shoot-tip explants showed the inflorescence colour and shape typical for the cultivars. Inflorescence colour of plants derived from adventitious buds were true-to-type in four cultivars: ‘Richmond’, ‘Lady Amber’, ‘Lady White’ and ‘Lady Yellow’. All plants of ‘Lady Apricot’ (originally: golden beet) and ‘Lady Salmon’ (salmon) propagated from adventitious buds technique showed altered inflorescence colour (respectively: purple gold; pink and white). ‘Lady Bronze’ (originally: reddish brown), ‘Lady Orange’ (orange brown) and ‘Lady Rosy’ (purple gold) propagated with adventitious buds had both typical and changed inflorescence colours (respectively: yellow; yellow and red; reddish pink). ‘Lady Vitroflora’ showed altered number of ligulate florets grown into tubes in inflorescence when propagated with shoot tips and leaves as explants. Those changes might be an effect of either chimeral structure or somaclonal variation of the plants investigated. The variation appears only if non-meristematical explants were used. The adventitious buds technique might be useful in chrysanthemum breeding as a source of a new variability.     

野生水芹组培快繁技术研究

以野生水芹茎尖、腋芽为外植体,研究了影响愈伤组织诱导和分化的激素浓度配比.结果表明,最佳外植体是茎尖;最佳愈伤组织和不定芽诱导培养基为MS 6-BA 4 mg/L,最佳继代增殖培养基为MS 6-BA 2 mg/L,最佳生根培养基为MS IBA 1 mg/L.并提出试管苗炼苗与移栽的最佳试验条件.     

美国芦荟茎尖和茎段离体培养植株再生研究

以美国库拉索芦荟（Aloe veraL.）茎尖和幼茎段为外植体,MS培养基配以不同浓度的NAA和6-BA进行离体组织培养。结果表明,以MS＋6-BA3 mg·L^-1＋NAA 0.5 mg·L^-1培养基对芽的诱导效果最佳,芽的诱导分化率最高,且试管苗健壮;增殖培养基以MS＋6-BA 2 mg·L^-1＋NAA 0.2 mg·L^-1效果最佳,有利于芽苗增殖;生根培养基用1/2 MS＋IBA 0.5 mg·L^-1＋1.5%蔗糖效果最好;芦荟外植体用0.1%升汞消毒时间以10 min为宜。通过研究采用一些措施有效地控制玻璃化苗的发生。     

西洋杜鹃组织培养的初步研究

以西洋杜鹃的两种不同花色品种‘红宝石'(R.Redjacj简称RR)、‘粉冠军'(R.Brittania简称RB)的无菌苗叶片和茎段(包含茎尖)为外植体,研究不定芽的诱导、壮苗及生根。结果表明,叶片诱导不定芽最佳培养基为:DJ+ZT2.0+NAA0.2+琼脂0.8%+蔗糖30g/L,‘红宝石'增殖倍数10.9,‘粉冠军'增殖倍数29.63。茎段(包含茎尖)增殖的最佳培养基为:DJ+ZT0.5+NAA0.05+琼脂0.8%+蔗糖30g/L,‘红宝石'增殖倍数为2,‘粉冠军'增殖倍数为3。生根最佳培养基为:Read+NAA0.2mg/L+AC3g/L,‘红宝石'平均生根率达84%,‘粉冠军'平均生根率达93%。     

Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.     

‘艾西丝’南瓜子叶的离体培养

用‘艾西丝’南瓜子叶作外植体离体培养,成功地建立了一套子叶高频率诱导再生芽的程序。在ＭＳ＋ＢＡ ４．０ｍｇ／Ｌ＋ＩＡＡ０．４ｍｇ／Ｌ的培养基上,以４ｄ苗龄子叶基部切块为外植体,其不定芽的诱导率最高,达５０％,在ＭＳ＋ＢＡ０．２５－０．５ｍｇ／Ｌ的培养基上继代,增殖培养效果较好,在无激素的１／２ＭＳ培养基上最易生根。     

甘蔗茎尖胚状体脱毒苗快繁技术研究

以甘蔗新台糖22号为材料,比较茎尖胚状体、茎尖与腋芽3种分化成苗繁育方法在不同时期接种、不同激素水平下的组培苗增殖速度、苗素质及脱毒效果。试验结果表明：组培苗繁殖速度以茎尖胚状体分化苗最快,增殖5代后扩繁2589倍,茎尖297倍,腋芽104倍,培养基以6-BA 1.5mg/L＋NAA 0.01-0.1mg/L增殖效果最好;组培苗质量与繁殖速度相反;出现不正常生长的苗类型有白化苗、细弱小苗、玻璃化苗、疯长苗4种,茎尖胚状体苗发生率1.77%,茎尖苗1.56%,腋芽苗0.31%;不同处理间组培苗生根及移栽成活率差异不显著,生根率茎尖胚状体苗75.3%、茎尖苗76.9%、腋芽苗76.6%,移栽成活率茎尖胚状体苗94.8%、茎尖苗95.4%、腋芽苗95.1%,生根培养基以NAA7.5mg/L＋ABA 2.5mg/L最好;去除RSD、花叶病方面,以茎尖胚状体苗最好,RSD去除率95%、花叶病去除率100%,茎尖苗RSD去除率70%、花叶病75%,腋芽苗未能去除RSD、花叶病。应用茎尖胚性细胞再生植株,脱毒效果好,繁殖速度快,可克服目前脱毒苗生产中试管苗扩繁量小、成本高的难题。     

利用甲基磺酸乙酯和枯萎病菌毒素诱变筛选香蕉抗毒素突变体

 以化学诱变剂甲基磺酸乙酯（EMS）和香蕉枯萎病菌（Fusarium oxysporum f. sp. cubense）4号生理小种的粗毒素为诱变剂,以香蕉愈伤组织和分化芽为筛选材料,通过多步筛选法,优化了筛选体系,并筛选出了抗香蕉枯萎病菌毒素的突变体。以体积百分比浓度为0.5%的EMS 分别处理香蕉愈伤组织和分化芽40 min,获得“半致死剂量”效应,对存活的香蕉愈伤组织和分化芽在依次递增的粗毒素浓度（5.0%→10.0%→12.5%）胁迫下进行定向筛选,获得了抗香蕉枯萎病菌毒素的愈伤组织块和分化芽,从抗病分化芽突变体获得了再生植株。对再生植株抗毒素的性能进行鉴定,其病情指数（24.72）显著低于对照植株（96.11）,抗毒素效果达到74.28%。     

Induction of novel variants through physical and chemical mutagenesis in Barbeton daisy (Gerbera jamesonii Hook.)

Summary

Gerbera (Gerbera jamesonii) is an attractive ornamental flower of high economic importance. The present investigation was aimed at generating novel flower colour variants in the gerbera cultivar ‘Harley’ through physical and chemical mutagenesis. In vitro-raised shoot cultures of gerbera, established from petiole explants, were exposed to different doses of γ-rays (1.5, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, or 30.0 Gy) using a Cobalt-60 source emitting 2.51 kGy h^–1. To induce mutations through chemical mutagenesis, different concentrations of ethylmethane sulphonate [EMS; 0.1, 0.2, 0.5, 0.8, or 1.0% (v/v)] were administered for 10 min or for 20 min. The LD50 values calculated for shoot survival and the induction of mutations were approx. 6.5 Gy for γ-rays and 0.65% (v/v) EMS for 10 min, or ≤ 0.1% (v/v) EMS for 20 min. Investigations revealed a negative correlation between mutagen dose and plant survival, both in vitro and after acclimatisation. Morphological variants showing changes in leaf shape, leaf size, scape length, flower diameter, and flower colour were obtained. Significantly, early flowering was induced in all mutated plants compared to non-mutated plants.The high frequencies of colour variants obtained using Bγ-rays, or the application of EMS to in vitro-raised shoot cultures could be an effective way to improve gerbera cultivars.     

Propagating palms in vitro with special emphasis on the date palm (Phoenix dactylifera L.)

Tissue-culture methods are described for the vegetative propagation of several palm species either through shoot tip culture or plantlet differentiation via embryogenic callus. The influence of explant size, medium composition and physical environment required for the establishment of palm shoot tips in vitro was determined. Date palm (Phoenix dactylifera L.) seedling shoot tips of various sizes were cultured in either liquid or agar modified Murashige and Skoog (MS) medium containing 0.0–1.0 mg 1^?1 α-naphthaleneacetic acid (NAA) and 0.0–15.0 mg 1^?1 benzyladenine or N⁶-(Δ²-isopentenyl) adenine (2iP) in order to enhance shoot growth and induce axillary budding. Satisfactory date palm shoot tip growth and proliferation was obtained from explants that were 3 mm in length, consisting of the apical meristem region and 2–5 adjacent leaf primordia. Optimum shoot tip development and axillary budding was obtained by initially establishing explants on an agar medium for 2 weeks, then transferring to a liquid medium. Shoot tips from several palm species were cultured on MS media containing 100 mg 1^?1 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg 1^?1 2iP and 3 g 1^?1 activated charcoal, or on MS medium containing 1 mg 1^?1 NAA and charcoal, to determine their morphogenetic responses in vitro. Shoot tips of Metroxylon sp., Phoenix canariensis Hort. ex. Chabaud., P. dactylifera ‘Khalasa’, ‘Thoory’ and ‘Zahidi’, and P. roebelenii O'Brien planted on medium with 2,4-D and 2iP initiated callus, asexual embryos and free-living plantlets after 4–8 months in culture. Shoot tips from Erythea edulis S. Wats., P. canariensis, P. dactylifera ‘Khalasa’, Thoory' and ‘Zahidi’, Washingtonia filifera Wendl. and W. robusta Wendl. cultured on medium containing NAA developed into plantlets with well-developed leaves and adventitious roots within 2–6 months from the time of planting. In some cases, cultured date palm shoot tips gave rise to axillary buds.     

华东葡萄白河-35-1器官离体再生体系建立的研究

以华东葡萄株系白河-35-1(Vitis pseudoreticulata clone Baihe-35-1)的茎段和叶片为材料,通过器官发生途径研究了不同激素及浓度处理组合对华东葡萄白河-35-1无菌苗茎段和叶片诱导不定芽的影响。结果表明,通过调整不同的激素浓度组合和选择不同的外植体可以成功诱导出不定芽。较低的激素浓度组合有利于外植体不定芽的再生。所选用的外植体中,茎段比叶片更易诱导出不定芽。诱导出不定芽的最适激素组合为TDZ1mg·L-1+NAA0.05mg·L-1,其中茎段不定芽诱导率为12.8%,叶片不定芽的诱导率为3.3%。将不定芽置于MS基本培养基+0.2mg·L-1IBA培养基上培养,获得了完整植株。     

Micrografting of almond (Prunus dulcis Mill.) cultivars “Ferragnes” and “Ferraduel”

The success of various in vitro micrografting techniques, establishment of the rootstock, size of the microscion, and the effects of culture medium on the grafted seedling development for almond cultivars “Ferragnes” and “Ferraduel” were studied. In vitro germinated wild almond seedlings developed from seeds were used as rootstocks. Shoot culture initiation was successfully achieved from the above almond cultivars by culturing mature shoot tips from forced nodal buds, about 3–5 mm, on 0.7 mg/L BA and 0.01 mg/L NAA containing a MS medium. The regenerated adventitious shoots from in vitro cultures were maintained and proliferated by sub-culturing on a fresh medium every three to 4 weeks. Regenerated shoot tips, which were micrografted onto in vitro seedlings, resulted in the restoration of shoot proliferation. The results indicated that the most successful method for the grafting of tested almond cultivars was slit micrografting. High levels of micrograft take were achieved with all ranges of scions (4–15 mm) obtained from the regenerated shoot tips. Slow growth and lack of axillary shoot development on the micrografts were noticeable when the micrografts were cultured on hormone-free germination medium. In vitro micrografted plantlets were successfully acclimatized and no problems were encountered with the establishment of micrografted plants in vivo. The developed technique has demonstrated a high potential for application in the micropropagation of almond cvs. “Ferragnes” and “Ferraduel” and thereby, represents a feasible method for the renewal of almond orchards in Turkey and elsewhere in the world.     

北海道黄杨茎尖组织培养初报

以北海道黄杨的茎尖为材料,探讨了不同培养条件、生长素浓度等因子对建立茎尖再生体系的影响。结果表明:北海道黄杨茎尖诱导最适培养基为6-BA+0.5 mg/L+NAA 0.1mg/L,最适分化培养基为MS+6-BA 1.0 mg/L+NAA 0.2 mg/L,且在前10 d采用暗培养,之后采取光照培养。     

菊苣子叶离体培养与植株再生的研究

以菊苣无菌苗的子叶为外植体,研究其在附加不同浓度NAA和6-BA的培养基上的分化情况.结果表明:在不同培养基上,菊苣子叶均能诱导出淡黄色的愈伤组织和分化形成不定芽,但不同基因型菊苣的出愈率和不定芽诱导存在明显差别.诱导Rossa di Verona tardiva selez prelude和Rossa di treviso菊苣愈伤组织形成和芽分化的最佳培养基为MS+NAA 0.5 mg/L+6-BA 2 mg/L;而诱导In miscuglio菊苣愈伤组织和芽分化的最佳培养基为MS+NAA 0.5 mg/L+6-BA 3 mg/L.     

新型试管花卉姬松高效再生体系的研究

 以新型试管花卉多肉类植物姬松（Crassula clavata N. E. Brown）为材料进行高效再生体系 的研究。结果表明：以姬松茎尖为外植体的初代培养中,MS 培养基添加IBA 并未诱导出根系生成,而是 直接诱导出丛生芽,在MS + IBA 0.3 mg · L-1 培养基上,由叶片表面诱导出的丛生芽质量最佳;在继代培 养中,最佳培养基为MS + NAA 0.1 mg · L-1 + 6-BA 0.5 mg · L-1,诱导产生大量胚性愈伤组织,且表面有 芽体分化;在彩色培养中,在1/2MS 中添加0.25 mg · L-1 的食用色素亮蓝,观赏效果佳且对植株生长无毒 害。     

Clonal propagation of mulberry (Morus indica L. cultivar M-5) through in vitro culture of nodal explants

A high frequency of sprouting (80.0%) and shoot differentiation was observed in the primary cultures of nodal explants of Morus indica L. cultivar M-5 on MS medium supplemented with 2,4-D (0.3 mg/l). In vitro proliferated shoots were multiplied rapidly by culture of shoot tips on MS medium with BAP (0.5 and 1.0 mg/l) which produced the greatest multiple shoot formation. Multiplication was also achieved by culture of shoot tips on MS medium with BAP (4.0 mg/l) and GA₃ (0.05 mg/l) which facilitated the elongation of shoots followed by sprouting of axillary buds of in vitro grown shoots. A high frequency of rooting (86.7%) with development of healthy roots was observed from shoots cultured on medium with 2,4-D (1.0 mg/l). Plants with well developed roots were transferred to soil with a survival frequency of 80%.     

Influence of cytokinins on in vitro morphogenesis in root cultures of Centaurium erythraea—Valuable medicinal plant

The purpose of this work was to acquire more information on the capacity of in vitro grown Centaurium erythraea Gillib. normal and hairy root cultures to simultaneously regenerate adventitious buds and to evaluate the variations induced on regeneration response by treatments with six cytokinins. Explants from normal and hairy root cultures were cultured on half-strength MS medium (1/2 MS) with kinetin (KIN), N⁶-benzylaminopurine (BA), 6-γ,γ-dimethylallylaminopurine (2IP), N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)urea (TDZ) and 6-[4-Hydroxy-3-methyl-but-2-enylamino]purine (ZEA), used alone in six different concentrations. The average number of adventitious buds per explant was promoted by all of cytokinin treatments. Urea-type cytokinins, TDZ and CPPU were more effective for the induction the morphogenesis of adventitious buds than adenine-type cytokinins. We found that the 1.0 μM CPPU induced the largest number (25.6, 18.2, respectively) of adventitious buds in normal and hairy root culture. TDZ-supplemented media induced highest number of adventitious buds (24.2) from normal root explant, but from hairy root explant average number of buds is lower (20.5). Regenerated shoots were excised and placed on 1/2 MS medium without hormone. The rooted plantlets were successfully acclimatized in greenhouse conditions.     

Effects of high vineyard temperatures on the grapevine leafroll associated virus elimination from Vitis vinifera L. cv. Napoleon tissue cultures

In vitro culture of shoot tips and axillary buds was used for virus elimination from the Spanish autochthonous table grapevine cultivar Napoleon which was infected by Grapevine leafroll associated virus-3 (GLRaV-3) and Grapevine fanleaf virus (GFLV). High percentages of GLRaV-3-free plants (91–100%) were obtained by establishing shoot tip cultures from infected mother plants of the 29-228, 74-16 and 77-266 clones. Lower percentages of virus-free plants (71–87%) were obtained by in vitro culture of the first, the second and the third axillary buds of the growing distal shoots. Percentages of virus-free plants obtained with both shoot tips and axillary buds varied according to the time of the year when the explants were collected and the bud position on the shoot. A increased efficiency of in vitro tissue culture methods was observed when cultures were established in summer and it was due in part to the high vineyard temperatures reached in the southeast of Spain during the hot season. GFLV- and GLRaV-3-free plants were only obtained by thermotherapy in combination with tissue culture methods from co-infected plants of the clone 39-29.     

茄子子叶下胚轴离体再生体系建立

以3份茄子材料无菌苗子叶和下胚轴为外植体,研究其在MS附加不同浓度6-BA和NAA的培养基上的分化情况.结果表明:在不同培养基上,茄子子叶和下胚轴均能诱导出愈伤组织和分化形成不定芽,但不定芽诱导率存在明显差别,诱导3份材料下胚轴愈伤组织形成和芽分化的最佳培养基均为:MS+6-BA 2.0 mg/L+NAA0.1 mg/L,子叶因品种不同有较大差异,紫色长棒茄芽分化最佳生长调节物质组合是6-BA 1.0 mg/L-+NAA 0.5 mg/L;紫红线茄是6-BA2.0 mg/L+NAA 0.1 mg/L;紫黑短棒茄是6-BA 3.0 mg/L-+NAA 0.5 mg/L.