Studies on the possible role of micropropagation in the dissemination of the strawberry crown rot pathogen Phytophthora cactorum

Summary

Attempts to develop a method for establishing latent infection by the crown rot pathogen (P. cactorum) in strawberry stolon tips or unrooted plantlets were largely unsuccessful. One month after dipping stolon tips in suspensions of zoospores (from 10 to 10³ ml^?1) 6% had died and 6% had become severely necrotic; only 2% of stolons with no obvious necrosis yielded P. cactorum in isolation on a selective medium. When young, pre-rooted plantlets were sprayed with more concentrated zoospore suspensions (from 10³ to 10⁵ ml^?1) and grown on for only one week, 9% died, 51% became necrotic and 40% remained apparently healthy; 22% of the “healthy” plantlets yielded P. cactorum. The frequency of isolation from different parts of the plantlets indicated that most infections originated in the base of the crown, probably via root initials. When 298 stolon tips and 357 meristems were excised from symptomless plantlets one week after inoculation with a suspension of 10⁴ zoospores per ml, P. cactorum grew conspicuously into the culture medium and killed the plant tissue in 6% of stolon tip cultures and in 0.8% of meristem cultures. Of 380 micropropagation cultures successfully established from explants after inoculation not one yielded the pathogen on destructive sampling although other microorganisms were detected. It is concluded that the crown rot fungus does not become cryptically established in micropropagation cultures of strawberry and that, conversely, the technique of micropropagation is a reliable way of ensuring freedom from this pathogen in plant material.     

Regeneration of Plants from Alginate-encapsulated Shoot Tips of Myrtle (Myrtus communis L.)

Myrtle growing naturally in the Mediterranean Region in Turkey, is among the economically important plant species. The present study emphasized on in vitro conservation of M. communis by encapsulating regenerated shoot tips. In this research we reported synthetic seed production and subsequent conversion of encapsulated shoot tips into plantlets comparing with nonencapsulated shoot tips for myrtle. Two different myrtle genotypes were used for synthetic seed production. Sodium alginate solution at the rate of 3.0% and 100?mM calcium chloride solution were prepared for encapsulation. Encapsulation was accomplished by mixing shoot tips into sodium alginate solution and dropping these in calcium chloride solution for 25–30?min. Encapsulated and nonencapsulated shoot tips were cultured in MS (Murashige and Skoog) media supplemented with different BAP (6-Benzylaminopurine) concentrations (0, 0.5, 1, 2?mg L^?1). After six weeks, all shoots were transferred to MS media containing 1?mg L^?1 IBA for in vitro rooting. As a result, the highest germination rate was obtained on the BAP-free MS media. The best BAP concentrations were detected as 0.5 and 1?mg L^?1 for micropropagation. Genetic stability of plants coming from encapsulated and nonencapsulated shoot tips was tested by ISSR markers. Based on the results, there were no genetic differences among the samples.

     

马铃薯茎尖小滴玻璃化法超低温保存及其再生植株的遗传稳定性

以马铃薯试管苗为试材,对其茎尖小滴玻璃化法超低温保存的影响因素进行了研究,并对再生植株进行了遗传稳定性检测。结果表明,马铃薯茎尖依次在含有0.3 mol · L-1和0.5 mol · L-1蔗糖的液体MS培养基中预培养各1 d后,在0 ℃下PVS2处理30 min,转到铝箔条上PVS2小滴上（约15 μL）,将粘有茎尖的铝箔条在液氮里蘸一下,然后直接装入盛满液氮的冷冻管中,投入液氮至少保持1 h。室温下用含有1.2 mol · L-1蔗糖的MS液体培养基解冻并洗涤30 min后,接种到MS + 0.5 mg · L-1 Zeatin + 0.1 mg · L-1 NAA＋1.0 mg · L-1 GA3恢复培养基上,存活率和再生率最高达79.91%和62.52%。通过SSR分子标记检测,再生植株的遗传稳定性没有发生改变。     

Influence of seed position within the fruit on seedling quality and in vitro shoot tip production of jackfruit

This study reports on the influence of seed position within the fruit and shoot development on the production of jackfruit in vitro. Seeds were extracted from the different sections in the fruit and shoot tips excised from the resultant seedlings. The tips were cultured in vitro on media supplemented with 6-benzylaminopurine (BAP) or thidiazuron (TDZ). The seeds from the middle and basal sections of the fruit were larger and produced larger seedlings than those from the top. Seeds from the basal position took 10 days to germinate and produced seedlings 11 cm tall with four or five leaves. Partially covered and fully open shoots from 6–8 week-old seedlings performed best. The highest frequency of shoot induction (100%) and multiple shoot production (7.31 ± 1.2) with a mean length of 3.6 ± 0.13 cm was observed on Murashige and Skoog (MS) media supplemented with 3.0 mg/L BAP. Driver and Kuniyuki (DKW), and MS media were equally effective in shoot multiplication. More than 80% of the rooted plants survived culture, indicating that this technology could be used to conserve wild germplasm.     

Effects of microbubble generation methods and dissolved oxygen concentrations on growth of Japanese mustard spinach in hydroponic culture

The use of microbubbles (MB) can improve the oxygen supply to plants grown under the deep flow technique of hydroponic culture. In a previous study, we compared the growth of komatsuna (Brassica rapa L. perviridis, Japanese mustard spinach) plants grown under hydroponic culture with MB generated by the pressurisation (P) method and the gas-water circulation (G) method. Plant growth was significantly lower in the presence of the P-MB than the G-MB. In this study, we aimed to identify the factor(s) responsible for the poor growth of komatsuna plants in the presence of P-MB. At three weeks after planting, the growth of the P-MB plants was less than the growth of G-MP plants and controls, regardless of dissolved oxygen concentrations. Analysis of the root tips by transmission electron microscopy showed plasmolysis of the P-MB root tip cells but not of the G-MB and control root tips. Our results suggest that the growth inhibition of plants grown in the presence of P-MB is due to inhibition of water and nutrition absorption from root tip cells due to this plasmolysis. This is likely due to oxidisation of root tip cells by hydroxyl radicals generated by many fine MB and/or osmotic stresses caused by the MB.     

Improved Culture of Apical Tissues for Production of Virus-Free Strawberries

Strawberry plants of fourteen varieties were raised from dissected apices of stolon tips and axillary buds excised at a length of less than 0·80 mm.

Two different media were used. On one of them 50% of tips from non-heat-treated plants developed roots and of these 54% survived to maturity; on the other medium (White’s augmented with coconut milk and sucrose) only 35% developed roots but 80%of these rooted tips grew to mature plants.

Tips excised from heat-treated plants grew more rapidly, and a higher proportion reached maturity, than those grown from untreated plants.

The smaller tips (<0·4 mm.) rooted less readily than larger ones (0·4–0·8 mm.) and grew more slowly to a size suitable for potting.

The plants of nine varieties were tested for virus infection by grafting to Fragaria vesca and F. virginiana 12–18 months after excision. One variety was freed from yellow edge (virus 2), three varieties from vein chlorosis (virus 4), two from crinkle (virus 3), and two from latent A virus.     

Aseptic micropropagation of Rhododendron P.J.M. hybrids

Summary

Shoot tips established in vitro better than single nodes. Five-cm shoot tips gave better Stage I results than 2-cm shoot tips. The best microshoot proliferation rates were obtained with isopentenyladenine (2iP) at 5 and 10 mg l^?1; tetrahydropyranyl-benzyladenine was ineffective. Microshoots rooted well, regardless of the level of 2iP in the Stage II medium. Stem-cutting-macropropagated plants generally were more variable in height, had less basal branching and were more erect than plants from microculture.     

Cryopreservation of in vitro almond shoot tips by vitrification

Summary

Shoot tips of two almond scion cultivars, ‘Ne Plus Ultra’ and ‘Nonpareil 15-1’, and one almond/peach hybrid rootstock were successfully cryopreserved using a one-step vitrification technique. Three week old in vitro cultures were cold-hardened at 4°C on the multiplication medium (Murashige and Skoog for ‘Ne Plus Ultra’ and the hybrid rootstock; Almehdi and Parfitt for ‘Nonpareil 15-1’) for three weeks. Shoot tips, 2–2.5 mm long, were excised and precultured for 1 d at 4°C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25°C for 45 min for the almond scion cultivars and 60 min for the hybrid rootstock, and then stored under liquid nitrogen (LN) for at least 3 d. After rapid thawing at 30°C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH₄NO₃ or (NH₄)₂SO₄. Shoot regeneration was usually observed within 2–3 weeks. Survival after LN, recorded as the percentage of shoot tips that produced at least one new shoot four weeks after thawing, was 87.5, 60.0 and 72.5% for ‘Ne Plus Ultra’, ‘Nonpareil 15–1’ and the hybrid rootstock respectively. The one-step vitrification method is a promising simple technique for cryopreserving almond scion and rootstock shoot tips from in vitro cultures.     

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。     

Biochemical and morphometric analysis of Rosa tomentosa and Rosa rubiginosa during application of liquid culture systems for in vitro shoot production

Two liquid culture systems, rotary shaker (RS) and temporary immersion system (TIS) for the propagation of Rosa rubiginosa and Rosa tomentosa were investigated and compared with solid medium culture. Shoot tips were grown on basic Murashige and Skoog medium with 20 mg dm^?3 Fe EDDHA, 1 µM BA, 1.5 µM GA₃ and 3% sucrose for six weeks. Of the different culture systems, liquid cultures resulted in better growth and development of roses. Both RS and TIS cultures improved biomass growth, multiplication rate, leaf blade area and plant height; however, RS reduced the shoot dry mass content. There were no changes in the content of chlorophyll a, b, a + b in plants developed in the tested systems. Use of a solid medium favoured the accumulation of phenolic compounds, soluble sugars, and carotenoids, but it was accompanied by browning and necrosis of shoots. Plants propagated in TIS were characterised by the high content of phenolic compounds and soluble sugars, but they had the highest multiplication rate. Use of TIS resulted in eight times the number of shoots for R. tomentosa and twice as many for R. rubiginosa compared to solid medium.     

Tissue-culture propagation of banana

The possibility of clonal propagation of banana through tissue culture was explored. Shoot tips isolated from the rhizomes were found to be suitable material for plantlet production in vitro. Excised shoot tips with the youngest leaves produced only one plantlet. However, shoot tips with several older sheathing leaf bases enclosing the axilary buds regenerated multiple plantlets. Individual shootlets, when separated and sub-cultured, produced a new crop of multiple shoots. The plantlets obtained from both types of explants have been successfully transplanted to soil and grown to maturity.     

Direct somatic embryogenesis from explants obtained from in vitro germinated embryonic axes of Camellia sinensis (L.) O. Kuntze

Summary

Studies on direct somatic embryogenesis in several types of explant from in vitro plantlets of tea cultivar TRI 2025 were undertaken to select those most suitable to induce cotyledonary-type somatic embryos. Mature zygotic embryonic axes were surface-sterilised and cultured on MS medium without growth regulators containing 0.6% (w/v) agar. Results showed that 65% of embryonic axes that converted into plantlets at the fifth week of culture had succulent leaves. Several types of explant (normal leaves, large and small succulent leaves, hypocotyl segments and root tips) were isolated from in vitro plantlets at the fifth week and cultured on half-strength MS medium containing 2 mg l^–1 6-benzylaminopurine (BAP) and 0.2 mg l^–1 naphthalene acetic acid (NAA). Morphological and histological observations on somatic embryogenesis were made. The results indicated that somatic embryos were produced at high frequency (25 – 50%) directly from the surface of hypocotyl segments (HS) and large succulent leaves (LSL) after 6 weeks of culture. Efficient somatic embryogenesis was induced in small succulent leaves (SSL) after 16 weeks. Most somatic embryos originated directly from the cortical tissues of HS or the upper epidermal layers of SSL or LSL. HS and SSL from in vitro plantlets gave the highest production of typical, firm somatic embryos for use in tea improvement programmes and for in vitro conservation of tea germplasm.     

The effects of alternatives to soil residual herbicides on weed control,yield and quality of hops

Summary

Several non-chemical methods of weed control in hops (Humulus lupulus) were evaluated in a three-year study. The results show that the use of cultivation, grass and winter rye cover crops, straw and plastic mulches all gave satisfactory weed control in both alleyways and hop rows. Data are presented on the effects of the main treatment groups, on the yield, alpha-acid content and alpha yield of hops. The effects on other hop cone quality criteria are also reported. Cultivation to control weeds had to be timely, and by the end of the study, numbers of weeds germinating in the spring were increasing. The use of a winter rye cover crop was successful in reducing over-wintering weed populations in cultivated plots. Grass reduced yield but raised the alpha-acid content of the cones, resulting in no overall loss in yield of alpha-acid. Yield reductions were overcome by adding extra nitrogen as an early spring top dressing, but this tended to reduce alpha-acid content to give similar alpha yields. Analysis of soil mineral nitrogen contents of soils to 90 cm depth in spring and autumn indicated that extra spring nitrogen was readily taken up by grass swards, helping to overcome its competitive effect on the hop plants. Winter rye reduced soil mineral nitrogen levels although this failed to reach statistical significance (P>0.05) but this nitrogen was returned to the soil when the cover was cultivated out in spring. Straw mulches tended to raise alpha-acid levels enough to raise alpha yield one year by 5% compared with conventional residual herbicide, although this failed to reach statistical significance (P>0.05). The implications of the results to growers of hops are discussed.     

In vitro regeneration and Agrobacterium mediated transformation in gladiolus

Summary

In vitro regeneration and transformation studies were conducted on two cultivars of gladiolus. Cormels of 1.0 to 1.5 cm diameter cut into 2–3 mm thick slices of top, middle and bottom, and in vitro derived bisected shoot tips were used as explants on MS medium supplemented with 18.6 μM kinetin for multiple shoot induction. Amongst the cormel slices, the top slice gave better shoot induction response of 89% with an average of 2.4 shoots per explant over both cultivars. In vitro derived bisected shoot tips were inoculated on the medium oriented cut-side up, cut-side down and vertically both with and without the cormel base attached. Bisected shoot tips without attached cormel base and inoculated in the cut-side down orientation showed an average of 90% shooting response. In vitro derived shoot tips were used as explants for transformation. Explants were wounded by scalpel and particle bombardment with 1.6 μm naked gold particles by the biolistic delivery system. The wounded explants, after 3 d of recovery period, were co-cultivated with Agrobacterium strain LBA4404 harbouring the binary vectors pBI141 and pTOK233 which contained gus reporter gene with rice actin and 35S promoters respectively. GUS expression frequencies of 5.3% and 23% was obtained from scalpel and particle bombardment wounded explants, respectively. Particle wounded explants showed an average of 63 and 103 GUS spots when co-cultivated with pBI141 and pTOK233 binary vectors respectively. Explants co-cultivated with pBI141, after three weeks of selection on antibiotic containing medium showed blue streaks of GUS expression. It was concluded that Agrobacterium could infect the monocot gladiolus and transform the tissue eficiently when tissues were prewounded with naked gold particles delivered by particle gun.     

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 %～23. 7 % , 嫩梢上的茎尖成苗率为27. 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。     

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.     

嘉定白蒜脱毒微鳞茎诱导的快繁技术研究

以嘉定白蒜为试材,研究了不同茎尖脱毒方法与不同外植体启动培养基对嘉定白蒜无菌苗建立与脱毒率的影响、不 同增殖培养基与切割方式对潜伏芽激活与增殖的影响,以及组培苗分株等级标准与培养基配方对诱导组培微鳞茎形成的影 响。结果显示：带1 个叶原基的茎尖,在MS+6-BA 1.0 mg·L^-1+NAA 0.1 mg·L^-1 的培养基上成苗率高,达76%;采用单株 假茎去顶后,取其基部1.0 cm 假茎茎段,再沿中轴线对半纵切的方式,在MS+6-BA 1.0 mg·L^-1+NAA 0.10 mg·L^-1 的培养基 上能较好地激活叶腋潜伏芽,增殖倍数达3.22;1 个叶原基包裹的芽原基茎尖脱毒成效最好,达100%;假茎茎粗3 mm 的组 培苗单株,在MS+NAA 0.1 mg·L^-1+ 糖90 g·L^-1、pH 为7.0 的培养基中微鳞茎诱导率最高,达94.8%。茎尖脱毒方式、叶腋 潜伏芽激活切割方式和组培微鳞茎诱导的培养基配方,是嘉定白蒜脱毒与快繁的技术关键。     

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.     

激素、光照对"红脸颊"草莓茎尖分化的影响

以草莓品种红脸颊茎尖为外植体,研究了激素、暗处理、光照强度对茎尖分化的影响,以期建立高效的茎尖分化体系。结果表明,在MS基本培养基中,添加6-BA0.5mg/L＋NAA0.1mg/L的激素配比是诱导＂红脸颊＂草莓茎尖分化成苗的最佳激素组合;暗处理5d后采用2000lx光照光培养（14h/d）的条件最有利茎尖分化成苗,萌芽率86.7%。     

Production of four commercially cultivated Echinacea species by different methods of in vitro regeneration

Summary

Efficient in vitro procedures for mass propagation of four commercially important Echinacea species have been deveoped. Plants of E. angustifolia, E. pallida, E. paradoxa and E. purpurea were regenerated by three methods, namely axillary bud proliferation, adventitious shoot formation and somatic embyrogenesis. Shoot tips obtained from in vitro germinated seedlings, adventitious shoots or somatic embryo-derived plantlets, when cultured on Murashige and Skoog medium enriched with 1 μM 6-benzylaminopurine, 2 μM kinetin, 0.5 μM indole-3-butyric acid and 4 mg^–1 paclobutrazol multiplied three-fold within 3–4 weeks in culture. Incorporation of paclobutrazol in the shoot multiplication medium was necessary to recover healthy and robust shoots suitable for rooting. Direct, high-frequency shoot formation on intact leaves of shoots grown on 6-benzylaminopurine and kinetin-supplemented media, an unusual and novel observation made in this study, occurred in all the species studied. Rooting of in vitro developed shoots was achieved relatively easily with Murashige and Skoog basal medium rather than with auxin-enriched media. Culturing of hypocotyl explants on medium containing 3,6-dichloro-o-anisic acid (commonly known as dicamba), or 2,4-dichlorophenoxyacetic acid, resulted in direct somatic embryogenesis in all the species examined. The presence of cytokinin was required for somatic embryo germination, but further development of germinated somatic embryos into normal plantlets occurred in Murashige and Skoog medium. We conclude that the procedures described here could be used for rapid propagation as well as genetic transformation of commerically cultivated Echinacea species.