Influence of explant source on in vitro axillary shoot formation in oak seedlings

In vitro shoot development was slower for apical shoot explants of young oak (Quercus robur L.) seedlings growing on Woody Plant Medium containing activated charcoal than for nodal shoot explants. The rate of in vitro shoot development was slowest in explants taken from seedlings that were undergoing rapid shoot elongation and most rapid in explants taken from seedlings that had stopped elongating and had fully expanded leaves. Maximum rooting was achieved on half-strength Woody Plant Medium containing activated charcoal. Rooting ability was not influenced by explant source.     

In vitro regeneration of plantlets from embryonic and seedling explants of Engelmann spruce (Picea engelmannii Parry)

A protocol has been developed for the in vitro production of plantlets of Engelmann spruce. Embryos and various parts of Engelmann spruce seedlings formed multiple shoots when cultured on defined media containing a cytokinin. The site and time of occurrence of the shoot buds, as well as their number, differed in the various explants. The frequency of shoot-forming explants was influenced by the salt formulation used, the type and concentrations of cytokinins and their mode of application. Development of buds was achieved by transferring the explants to basal medium containing no growth regulators. Elongation of shoots was stimulated by reducing the concentration of salts and sugar, addition of activated charcoal and transferral to increased photoperiod and lower temperature regimes. Maximum rooting was induced by giving a pulse of high concentration of indolebutyric acid to the shoots. The roots developed within 8-10 weeks and the regenerated plantlets were transferred to soil under non-sterile conditions.     

Establishment of a high-frequency regeneration system in <Emphasis Type="Italic">Cerasus humilis</Emphasis>, an important economic shrub

Cerasus humilis is a species of small, perennial, drought-resistant and multipurpose deciduous shrub grown in arid and semi-arid conditions in northern China. In this study, an efficient protocol for the rapid micropropagation of C. humilis has been standardized using stem and/or leaf explants. Direct multiple shoot induction was observed when the stem explants were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The highest shoot induction was obtained when stem explants from adult trees were cultured on MS medium supplemented with 2.0 mg L^?1 6-benzyladenine (6-BA) and 0.9 mg L^?1 α-naphthaleneacetic acid (NAA). The leaf and stem explants cultured on MS medium with 1.0 mg L^?1 6-BA and 0.6 mg L^?1 NAA, and 0.5 mg L^?1 6-BA and 0.8 mg L^?1 NAA, respectively, produced the highest induction frequency of callus. Maximum proliferation of callus was observed on MS medium containing a combination of 0.5 mg L^?1 6-BA with 0.6 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-d). Optimal shoots differentiated from callus were obtained on MS medium supplemented with 5.0mg L^?1 6-BA and 0.9 mg L^?1 NAA. In vitro rooting was achieved on half-strength (1/2) MS medium containing 0.5 mg L^?1 NAA. Rooted plantlets were hardened under control conditions and successfully acclimatized under field conditions.     

Direct shoot bud regeneration from shoot tip explants of <Emphasis Type="Italic">Enicostema axillare</Emphasis>: an important medicinal plant

An efficient protocol has been developed for in vitro propagation of Enicostema axillare using shoot tip explants. The shoot tip explants were cultured on MS medium supplemented with various combinations of (BAP, KIN) and (NAA/IAA & IBA) in different concentrations between 0.5 and 2.0 mg/l for multiple shoot bud induction. The highest percent of (98.51 %) was observed at 1.0 mg/l BAP in combination with 0.2 mg/l KIN while maximum number of shoot buds (8.41 shoots/explant) was noticed on MS medium containing 1.0 mg/l BAP and 0.2 mg/l KIN combination. The highest frequency (90.82 %) of multiple shoot bud regeneration was observed at 1.0 mg/l BAP and 0.5 mg/l IBA with 15.12 ± 2.12 shoots/explants. The regenerated multiple shoots were transferred to half-strength MS medium augmented with different concentration of 0.5–2.5 mg/l IBA for rooting. Among the different concentrations of IBA tested, maximum percentage of rooting (100 %) was observed in MS medium augmented with 1.5 mg/l IBA. The rooted plantlets were successfully transferred into plastic cups containing soil and sand in the ratio of 1:1. Subsequently established in the field conditions with 90 % of survival rate. The protocol developed can be utilized for both large scale plant production and conservation of germplasm of this species. The described method can be successfully employed for large-scale multiplication and in vitro conservation as well as production of secondary metabolites of E. axillare.     

Shoot multiplication of Syringa reticulata var. mandshurica from in vitro cultured seedlings

We developed a shoot multiplication protocol for Syringa reticulata Blume var. mandshurica Hara from in vitro cultured seedlings that derived from in vitro germinated seeds. The shoots could be induced on Murashige and Skoog(MS) medium with proper plant growth regulator combinations of 6-benzylaminopurine(BA) and indole-3-butyric acid(IBA). The better medium for shoot multiplication and growth was MS + 5 mg L~(-1)BA +0.5 mg L~(-1)IBA + 20 g L~(-1)sucrose +7 g L~(-1)agar, and the corresponding shoot induction rate was 75 %. The plantlets grew well after rooting on 1/2MS medium(macro-elements of MS medium are at half-strength) supplemented with 1 mg L~(-1)IBA, and the survival percentage was 80 % at 16 weeks after transplanting.     

In vitro regeneration and the antioxidant enzymatic system on acclimatization of micropropagated Vitex trifolia L.

The objective of the study was to develop an in vitro shoot regeneration protocol by utilising shoot tips explant from Vitex trifolia L. Shoot tip explants obtained from a 3-year old plant was cultured on Murashige and Skoog (MS) medium supplemented with various concentrations (1.0, 2.5, 5.0, 7.5 or 10.0 µM) of thidiazuron (TDZ). The optimal level of TDZ supplementation to the culture medium was 5.0 μM for 15 d induction period. The highest number of shoots (22.2 ± 0.1) and shoot length (5.1 ± 0.1 cm) were achieved when TDZ-exposed explants were sub-cultured on MS medium containing 6-benzyladenine (1.0 µM) and 0.5 µM α-naphthalene acetic acid (NAA) after 8 weeks of culture. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5 µM NAA. During the acclimatization period, changes in activities of antioxidant enzymes were observed. Superoxide dismutase activity increased reaching maximum at 28th day after transplantation. Likewise, an upregulation of the catalase, ascorbate peroxidase and glutathione reductase enzyme activities were also observed. These observed changes reflected the ability of plants in developing an antioxidant enzymatic defence system aiding in survival against oxidative stress and in reducing release of free radicals. Plantlets were successfully hardened off and acclimatized in earthen pots containing garden soil with a survival rate of 90 %.     

Micropropagation of red sanders (Pterocarpus santalinus L.) using mature nodal explants

In vitro clonal multiplication of Pterocarpus santalinus L. was achieved using mature nodal explants of a 10-year-old elite quality tree. Combinations of serial transfer technique and incorporation of antioxidants (250 mg/l L-ascorbic acid and 50 mg/l citric acid) into the culture medium helped to minimize medium browning and improve explant survival during shoot sprouting. About 70% of explants were sprouted on Murashige and Skoog (MS) liquid medium containing 4.4 μM 6-benzyladenine (BA). The explant harvest period also influenced the bud break and shoot sprouting in nodal explants. The combination of 4.4 μM BA and 2.2 μM thidiazuron (TDZ) was found to be the most suitable growth regulator for obtaining the highest percentage of nodal segment sprouting (74%–75%), the number of secondary shoots per primary shoot (two or three), the shoot length (5–6 cm), the number of new nodal segments generated per active explant (four or five), and the multiplication coefficient (3.5) within 6 weeks. Repeated subculturing of nodal explants obtained from shoot cultures enabled continuous production of healthy axillary shoots. At the end of the sixth passage, about 90% of nodal explants produced five or six healthy green shoots, each being about 6.6 cm long with six or seven nodes. Multiplication coefficient was also increased from the first subculture (5.4) to the sixth subculture (8.3). The best rooting response was achieved on solidified half-strength MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 70% of the micropropagated plantlets were established successfully in 20-cm pots containing a mixture of soil and farmyard manure (4 : 1 ratio) and formed new leaflets.     

孔雀草杂交F1代的植株再生

以孔雀草（Tagetes patula）子叶、下胚轴和叶片为外植体,通过器官直接发生途径诱导形成不定芽,探讨植物生长调节剂组合、AgNO3浓度、蔗糖浓度和外植体类型等因素对植株再生的影响,建立了再生体系。结果表明：MS＋6-BA 1.0 · L^- 1＋NAA 0.5 · L^- 1＋AgNO31.0 · L^- 1＋蔗糖40 g·L^-1培养基最适合不定芽的分化和增殖,子叶不定芽分化率达90%以上,平均每外植体分化不定芽数达5.3个。不定芽较适生根培养基为1/2MS＋IAA 0.2 · L^- 1＋NAA 0.1 · L^- 1,生根率达到90%。     

中金系列杨树的组培技术研究

中金系列杨树是杨柳科杨属白杨派植物,选择其腋芽（或顶芽）、嫩茎段、嫩叶片等为外植体,进行愈伤组织诱导试验、不定芽分化试验、生根试验,筛选出了适合植株分化、生根的培养基成分;对生根试管苗进行移栽基质配比试验,确定组培苗最佳生长环境。总结出适合中金系列杨树组培的技术流程,保证了杨树的遗传稳定性,降低了培养成本,加快了培养速度。     

In vitro plant regeneration system for <Emphasis Type="Italic">Cassia siamea</Emphasis> Lam., a leguminous tree of economic importance

A method for rapid in vitro propagation of Cassia siamea Lam. using cotyledonary node explants, excised from 14-day old aseptic seedlings, has been established. Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) singly or in combination with auxins was used for regeneration studies. Among the single treatment of three cytokinins BA at 1.0 μM was found to be optimum for direct shoot regeneration as it induced an average of 8.20 ± 0.66 shoots per explant. The regeneration frequency further enhanced with the application of auxin along with optimal BA concentration. The highest frequency for shoot regeneration (90%), the maximum number of shoots per explant (12.20 ± 0.73) and the maximum shoot length (6.40 ± 0.07) cm were obtained on the medium consisted of MS + 1.0 μM BA + 0.5 μM NAA. Successful in vitro rooting was induced from cut end of the microshoots when placed on half-strength MS + IBA (2.5 μM). The regenerated shoots with well developed root system were successfully acclimatized and established in pots containing sterilized garden soil and garden manure (1:1) and grown under greenhouse conditions with 85% survival rate.     

In vitro regeneration of plantlets from mature embryos of Pinus ayacahuite

A plantlet regeneration protocol was developed for Pinus ayacahuite var. ayacahuite (Ehrenb.). Embryos from mature seeds from ten provenances were cultured in a 16-h photoperiod for 3 days on a medium containing 30 mM sucrose and 0.7% agar. Cotyledons from these embryos were subcultured onto MCM medium (Bornman 1983) supplemented with 50 micro M N(6)-benzyladenine and 90 mM sucrose for 2 weeks. Bud development and shoot elongation were maximized by subculturing the explants on half strength AE medium (von Arnold and Ericksson 1981), supplemented with 60 mM sucrose and 0.05% activated charcoal every 30 days. Seed source had a significant effect on the responses of the embryos to the bud induction protocol. For the provenance with the best response to bud induction, about 79% of the cultured cotyledons formed buds, and each cotyledon formed a mean of 9.1 buds, so that about 70 shoots could be induced from each seed. The best rooting response (40% rooting) was obtained by treating the shoots for 8 h with 100 micro M naphthalene acetic acid.     

Stimulation of in vitro organogenesis from epicotyl explants and successive micropropagation round in Cassia angustifolia Vahl.: an important source of sennosides

This report describes a protocol for the in vitro shoot induction and plant regeneration from epicotyl explants of Cassia angustifolia on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), Kinetin and 2-iP (0.5–10.0 μM). MS medium supplemented with BA (5.0 μM) was the most effective in inducing adventitious shoots and growth. The highest rate of shoot multiplication was achieved on MS medium supplemented with BA (5.0 μM) and Indole-3-acetic acid (IAA, 1.0 μM). The nodal segments excised from the shoots regenerated from BA (5.0 μM) and IAA (1.0 μM) were used as explants for next three round of micropropagation. The number of shoots significantly increased at successive round of micropropagation. For rooting, MS medium supplemented with 2.0 μM indole-3-butyric acid proved to be better than that supplemented with IAA or α-naphthalene acetic acid. The in vitro raised plantlets with well developed shoot and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse. About 52 plants (85 %) survived out of 60 plants transferred in garden soil.     

茛艻花组织培养技术

以茛艻花顶芽为外植体进行离体培养,成功建立了快速繁殖技术体系,结果表明,不同激素及其浓度对其增殖及根的形成影响显著。各个阶段适宜培养基:(1)增殖培养基为MS+BA 3.0 mg/L+NAA 1.0 mg/L,培养30天,增殖率稳定为4.65;(2)壮苗培养基为MS+6-BA 0.2 mg/L+IBA 0.2 mg/L;(3)生根培养基为1/2MS+NAA4mg/L+蔗糖20 g/L时,生根率达95%。     

In vitro and ex vitro rooting of micropropagated shoots using three green ash (Fraxinus pennsylvanica) clones

Shoot multiplication using seedling materials was achieved by subculture on Murashige and Skoog salts with Gamborg's B5 vitamins (MSB5) medium containing a combination of 5 M 6-benzyladenine (BA), 5 M thidiazuron (TDZ), and 1 M indole-3-butyric acid (IBA) with three green ash (Fraxinus pennsylvanica) clones, SD1009 (South Dakota origin), SD2002 (South Dakota origin), and KA2018 (Kansas origin). Shoots were rooted using in vitro and ex vitro methods. For in vitro rooting studies, elongated shoots were transferred to rooting plugs supplied with liquid MSB5 medium containing a 3×3 factorial arrangement of two different auxins, -naphthaleneacetic acid (NAA) and IBA, at three concentrations (0, 5, and 10 M). The most effective treatment for in vitro root number, root length, and shoot height was 5 M IBA. The three clones also were tested for ex vitro rooting using a quick dip in 1 mM NAA, 1 mM IBA, or control (no auxin). The maximal ex vitro rooting response occurred when shoot explants of the three clones were dipped in 1 mM IBA. Significant clonal differences were noted in response to in vitro and ex vitro rooting treatments. Rooted plantlets were acclimated to the greenhouse.     

苹果抗寒矮化砧木71-3-150茎尖培养快繁体系建立

以苹果抗寒矮化砧木71-3-150的茎尖为外植体,通过研究培养基中不同植物生长调节剂配比对芽苗诱导、增殖和生根的影响,筛选出适合71-3-150砧木茎尖组培快繁的培养基配方。结果表明,以MS为基本培养基,附加蔗糖30 g／L、琼脂6 g／L,适宜启动培养的植物生长调节剂为6-BA 1.0 mg／L＋NAA 0.05 mg／L;适宜增殖培养的植物生长调节剂为6-BA 1.0 mg／L＋NAA 0.05 mg／L,增殖系数可达到每4周4.75倍。以1／2MS （蔗糖15 g／L,琼脂6 g／L）为基本培养基,适宜组培苗生根的植物生长调节剂为IAA 1.0 mg／L＋IBA 0.6 mg／L,生根率达100％,每株苗平均生根9.6条,移栽成活率达90％以上。     

Micropropagation of Vegetable Rennet (Withania coagulans [Stocks] Dunal)—A Critically Endangered Medicinal Plant

Withania coagulans (Stocks) Dunal (Solanaceae), popularly called vegetable rennet, is a critically endangered and highly valued medicinal plant. Overexploitation and reproductive failure forced the plant species toward the verge of complete extinction. We describe here the development of a simple, rapid, and cost effective in vitro micropropagation system for W. coagulans for mass-scale production of true-to-type plantlets using nodal shoot segments. Exactly 95.5 ± 0.34% explants responded within 8–10 days (d) and produced multiple shoot buds (4.1 ± 0.10 shoots of 2.95 ± 0.15 cm length) on 0.8% agar-gelled Murashige and Skoog's (MS) basal medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), and additives (100 mg L^?1 L-ascorbic acid, 25 mg L^?1 each citric acid, adenine sulphate, and L-arginine). The shoots in cultures were multiplied by repeated transfer on MS medium with 4.44 μM BAP, 0.57 μM IAA, and additives. Further cultures were multiplied on a large-scale through the subculturing of shoot clumps differentiated in vitro, on MS medium supplemented with 1.11 μM BAP, 0.57 μM IAA, and additives. Maximum number (19.1 ± 0.28) of healthy (6.15 ± 0.25 cm) and viable shoots differentiated on this medium. The microshoots were rooted both in vitro and ex vitro. Exactly 67.3 ± 1.01% microshoots rooted in vitro within 25–30 d on agar-gelled half-strength MS salts supplemented with 29.52 μM indole-3-butyric acid (IBA) and 200 mg L^?1 of activated charcoal (AC). Alternatively, 73.8 ± 0.65% cloned shoots rooted on sterile soilrite (soilless compost and soil conditioner) under ex vitro conditions after pulse treatment with 2.46 mM IBA for 300 s. The clones of W. coagulans were hardened in a greenhouse within 40–45 d by slow and gradual exposure of plantlets from high relative humidity (RH; 70–80%) and low (26 ± 2°C) temperature to low RH (40–50%) and high (34 ± 2°C) temperature. The hardened plantlets were transferred to soil and stored in agro-net house with more than 90% survival rate. Replacement of pure and laboratory grade sucrose with commercial grade sugar, use of less expensive commercial grade agar-agar in culture medium, higher rate of shoot proliferation, single step ex vitro rooting, and hardening of plantlets in the greenhouse are advantageous features of the protocol. The micropropagation protocol defined here is reproducible, easy to follow, and would be helpful in large-scale restoration programs through true-to-type mass-multiplication of W. coagulans.     

松塔景天组织培养与快速繁殖的研究

以松塔景天幼嫩叶片作为外植体,利用含有不同浓度配比植物生长调节剂的培养基分别进行愈伤组织的诱导、不定芽的诱导、生根培养及驯化移栽,建立了松塔景天组织培养和快速繁殖体系。试验结果表明：诱导愈伤组织的最适培养基为MS＋6-BA 0．5 mg/L ＋NAA 0．05mg/L ,愈伤组织诱导率达87．78％;诱导不定芽的最适培养基为MS＋6-BA 1．0mg/L＋ NAA0．1 mg/L ,不定芽诱导率达88．89％;组培苗最佳生根培养基为1/2 M S＋IB A 0．3 m g/L ,生根率达91．33％;组培苗的移栽成活率达84％。     

珍稀竹种巨龙竹组织培养研究

对巨龙竹幼年竹和成年竹材料采集、处理、试管引入、丛芽诱导、丛芽增殖和生根、再生植株炼苗等进行系统研究,采用正交设计筛选出各阶段的最佳培养基.结果表明:丛芽增殖倍数与BA用量成正比,但随着BA用量加大或长期在高浓度BA上培养易产生花芽,说明BA有使丛芽细胞从营养状态向生殖状态转变的作用;KT在丛芽高生长方面起主导作用,能使丛芽节间伸长,使细胞从生殖状态向营养状态转变;椰乳有改善丛芽或苗生长的作用.在诱导丛芽生根过程中,IBA起主导作用,配合少量NAA使用可得到生根良好、移栽成活率较高的再生小植株.巨龙竹成年竹组织培养与外植体采集部位、采集时间、采集地等相关.     

西蒙得木组织培养繁殖技术的研究

通过对西蒙得木〔Simmondsia chinensis(Link)〕组织培养大批量繁殖技术的研究,探索基本培养基、生长调节物质及蔗糖含量等对嫩梢增殖及生根的影响,结果表明:采用春季生长的1年生实生苗以及6年生高产的优株茎节作外植体,培养于含有ZT 1～3 mg/L或BA 1～3mg/L和NAA 0.01～0.1 mg/L的MS培养基中,经30～40天,几乎所有腋芽均能长成嫩梢,切取长达4～6 cm的嫩梢,培养于含有NAA的1/2MS培养基中,经1周暗培养,1个月后生根率达93％,小植株移栽于含砂土的基质中,保持湿润,获得成功。用幼苗及优株作外植体的培养效果无显著差异,说明此法不受遗传型的限制。     

尾叶桉的组织培养及植株再生

研究了以尾叶桉(Eucalyptusurophylla)优树(U6无性系)无菌苗的叶子和茎段作为外植体诱导愈伤组织、丛生芽发生以及植株再生的过程。通过多种生长调节剂不同浓度组合的对比试验,确定了U6快繁体系的最适宜培养条件:(1)愈伤组织诱导培养基:MS 1-2mg/L2,4-D;(2)芽增殖培养基:MS 0.5mg/L6-BA;(3)生根培养基:1/2MS 2.0mg/LNAA。