Micropropagation ofFagus sylvatica L.

Summary An in vitro shoot multiplication system was established from juvenileFagus sylvatica L. tissues, and plantlets were regenerated. Embryonic axes were excised from beech seeds and germinated in vitro on media supplemented with 6-benzyladenine (BA) to obtain plantlets with axillary shoots. Shoot multiplication was maintained by sequential subculture of axillary shoot tips and basal segments on Woody Plant Medium supplemented with 0.5 mg/liter BA+2 mg/liter zeatin+0.2 mg/liter naphthaleneacetic acid (NAA). The effeciency of shoot multiplication clearly depended on the kind of explant used. Transfer to fresh medium every 2 wk during the 6-wk multiplication cycle improved multiplication rates. In the rooting stage, an initial 7-day dark period significantly improved rooting capacity and accelerated the emergence of roots on auxin-treated shoots. Adventitious buds were induced on the intact hypocotyls of the whole plantlets derived from the initial embryonic axis explants, especially on those cultured on medium with 1 mg/liter BA. Cotyledon and hypocotyl segments isolated from seedlings grown in vitro from embryos also exhibited capacity for adventitious bud formation, especially when cultured on media supplemented with 0.5 mg/liter BA + 0.1 mg/liter NAA.     

沙棘组织培养技术的研究

本试验选用中国沙棘的种子和休眠枝条作为材料,研究沙棘不同外植体的离体培养技术,试验结果表明,沙棘的无菌苗子与休眠枝条上的休眠芽都可以作为离体培养的良好外植体,沙棘的最适分化培养基是：1／4MS＋6－BA0．30mg/L NAA0．002mg/L,增殖,壮芽培养基是：1／4 MS＋6－BA 0．1mg/L NAA 0.004mg/L;最适生根培养基是：1／4MS＋NAA0.05mg/L iBA 0.2mg/L;愈伤组织诱导培养基是：1／4MS＋2,4－D 0．3mg/L。     

Rapid clonal propagation of Saccharum officinarum L. Vars. CO-6907 and CO-86249 and to assess the genetic uniformity through molecular markers

Abstract

An efficient protocol was developed for in vitro clonal propagation of Saccharum officinarum Vars. CO-6907 and CO-86249 through axillary meristem culture. Maximum meristem elongation was achieved on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kn) within 15 days of culture. Multiple shoots were induced from meristems on MS basal medium supplemented with 1.0 mg/L BA, 0.5 mg/L Kn, 0.25 mg/L 1-napthaleneacetic acid (NAA) and 3% (w/v) sucrose. Addition of 0.1–0.25 mg/L gibberellic acid into the multiplication medium found the better shoot elongation. Repeated subculture on multiplication medium induces higher rate of shoot multiplication. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 1.0–2.0 mg/L NAA or indole-3-butyric acid and 6% (w/v) sucrose. While either decreasing or increasing of sucrose concentration in the rooting medium, the percentage of rooting was reduced. Maximum percentage of rooting was achieved on medium having 2.0 mg/L NAA with 6% (w/v) sucrose. About 80% of micropropagated plantlets were hardened in the greenhouse and successfully established in the soil. Random Amplified Polymorphic DNA marker was used to detect the variability among the micropropagated plants developed through in vitro. The results showed that there was no polymorphism among the micropropagated plants. This study will help for propagation of quality planting material of high-yielding variety of sugarcane for commercialization.     

One step shoot tip multiplication and rooting of Digitalis thapsi L.

Shoot tips from seedlings of Digitalis thapsi L. were cultured on Murashige and Skoog's medium and the effect of various auxins (2,4-D, NAA and IAA) were analyzed alone or in combination with cytokinis (BA and kinetin). Shoot multiplication and direct rooting of the new shoots were obtained after four weeks of culture in MS medium without hormones, but callus formation and the appearance of abnormal phenotypes were frequent. The addition of auxins to the cultures prevented the formation of callus but not the appearance of variant phenotypes. Both drawbacks could be avoided by combination of NAA or IAA with BA or kinetin. The best results for shoot multiplication and direct rooting were obtained with 0.5 mg l^-1 NAA and 0.1 or 0.5 mg l^-1 kinetin.Abbreviations BA 6-benciladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - NAA naphtalene acetic acid - MS Murashige and Skoog     

罗布麻离体培养及快繁技术的研究

采用野生罗布麻(Apocynum venetamL.)顶芽及芽下茎段为外植体进行离体培养及快繁技术优化的研究。结果表明:适宜罗布麻离体培养的基本培养基为MS,适宜外植体起始分化的培养基为:MS BA 1.8mg/L KT 0.5mg/L,分化率为88.9%以上;适宜茎段增殖的培养基为MS BA 2.0mg/L KT 0.5mg/L,繁殖系数最高达5.67倍,通过切段繁殖还可提高繁殖系数3倍;适宜生根的培养基为MS IBA 0.5mg/L NAA 0.02mg/L,生根率达90%以上。     

转抗虫基因欧美黑杨离体快繁技术研究

以抗虫欧美黑杨的叶,带腋芽茎段为外植体进行离体快繁技术研究。最佳接种时间为8月份,新芽生长迅速。基本培养基为MS,较适初培养基为MS＋6－BA0.5mg/L（以下单位同）＋NAA0.01mg/,附加30g/L,蔗糖,7g/L琼脂。愈伤组织诱导并同时分化出新芽培养基为MS＋6－BA1.5 NAA0.3,附加40g/L蔗糖,6g/L琼脂。继代增殖培养基为MS 6-BA1.0 NAA0.1 GA2.0,附加30g/L蔗糖,5g/L琼脂。生根培养基为MS＋IBA2．0。     

In Vitro Multiplication of Beta Vulgaris L. throughout Excised Shoot Tips

Friable calli were induced from mature excised shoots of Bambusa vulgaris on Murashige and Skoog's (MS) medium supplemented with 2.2 μM6-benzylamino-purine (BAP), 9.04 μM 2,4-dichlorophenoxyacetic acid and 14.76 μM indole-3-butyric acid (IBA) with 3 % (m/v) saccharose. Adventitious shoots with root hairs were achieved from calli on MS medium supplemented with 13.33 μM BAP and 1.23 - 2.46 μM IBA within 4 weeks of subculture. The frequency of shoot bud regeneration was better in the light incubated cultures than in the dark incubated cultures. Isolated shoots were rooted on liquid half-strength MS basal medium supplemented with 0.49 μM IBA and 2 % (m/v) saccharose. Histological observations confirmed the regeneration of shoot buds from calli. The rooted plantlets were successfully transferred to greenhouse. This revised version was published online in July 2006 with corrections to the Cover Date.     

无融合生殖油菜AMR—1花托离体培养的研究

报道了不同激素浓度对无融合生殖没菜花托器官分化效果的研究,结果显示：（１）以ＭＳ为基本培养基,以带有子房和花柄的花托为外植体离体培养,花托、花柄切口部位直接芽诱导的最佳激素配比为４．０ｍｇ／Ｌ６－ＢＡ＋０．０１ｍｇ／Ｌ ＮＡＡ,频率为５８．８２％,花托、花柄部位先形成愈伤组织,继而分化出丛生芽的最佳激素配比为５．０ｍｇ／Ｌ ６－ＢＡ＋０．５ｍｇ／Ｌ ＮＡＡ,频率为８４．００％;（２）腋芽增殖的最佳     

Micropropagation of Dendrocalamus asper by shoot proliferation using seeds

An efficient and reproducible procedure for the large-scale propagation of Dendrocalamus asper is described. High-frequency direct shoot proliferation was induced in aseptic seed cultures of D. asper on modified Murashige and Skoog's (1962) medium supplemented with 1.0–10.0 mg/l benzyladenine (BA). Multiple shoots (1–25) were formed within 5 weeks of seed culture without root formation. The shoot-forming capacity of seeds was influenced by the BA concentration in the medium. Proliferating shoot cultures were established by repeatedly subculturing shoots in propagules of 3 shoots each. A multiplication rate of 15–16 fold was achieved on MS medium +3.0 mg/l BA. Roots were formed on excised propagules of 3–5 shoots when transferred to MS medium containing 10.0 mg/l indole-3-butyric acid (IBA) or 3.0 mg/l 1-naphthaleneacetic acid (NAA). Plantlets were hardened, acclimatized and established in soil, where they exhibited normal growth. Received: 10 April 1998 / Revision received: 18 November 1998 / Accepted: 12 December 1998     

In vitro root induction in axillary microshoots of Quercus robur L.

Whilst considerable efforts have been made to optimise shoot multiplication and rooting in oak, little attention has been paid to the impact of conditions used for multiplication on subsequent root formation. An optimised technique for rooting of oak microshoots has been developed to assess the effect of cytokinin treatments applied to shoot multiplication cultures on the subsequent rooting of microshoots. We found IBA to be more effective at inducing root formation in microshoots than NAA. Efficient rooting of oak microshoots (80%) was achieved after 35 days on medium supplemented with 1.0 mg litre^-1 IBA. Lower concentrations of IBA reduced the frequency of root formation and significantly increased the time taken for microshoots to form roots. High concentrations of IBA (3.0 mg litre^-1) produced similar rooting frequencies but with significantly increased numbers of roots formed by each microshoot. However, high concentrations of IBA stimulated the production of basal callus. Rooting of microshoots was unaffected by the concentration of BA used during shoot multiplication, although basal callusing was greater in microshoots taken from multiplication medium supplemented with the highest concentration of BA (1.0 mg litre^-1) and rooted on medium supplemented with 3.0 mg litre IBA. Reducing the period of exposure to auxin to 7 days by transferring microshoots to auxin-free medium increased the frequency of root formation (84%), led to more rapid root formation and a reduction in basal callus formation.     

大叶种胡椒实生苗茎尖培养和合子胚培养研究

利用各种表面消毒方法对采自海南岛三个地区的胡椒大田植株的外植体进行消毒试验,由于内源性污染,除胡椒成熟种子外,其它各种大田外植体的表面消毒均未能成功。以胡椒成熟种子无菌萌发的实生苗茎尖作外植体,在1/2MS(MS或B5)+1.5mg/LBA+0～0.2mg/LIAA(或NAA)上可实现丛生芽增殖。茎尖水平或竖直接种方法显著影响茎尖的增殖;水平接种茎尖的生长和增殖效果优于竖直茎尖接种方式。茎尖增殖率随BA浓度的增加而提高,但BA浓度大于2.0mg/L时会使苗芽的质量降低,愈伤组织产生严重,苗芽细小,抽出不明显,颜色发黄甚至变白。附加或不附加100mg/LAdSO4对丛生芽增殖没有明显影响。生根培养基以1/2MS+1.0mg/LIBA+0.5～1.0mg/LIAA为最优,生根率可达100%;在细沙∶土∶椰糠(1∶1∶1)的基质中常规炼苗,成活率可达98%以上。液体纸桥法对胡椒种胚进行培养,在不附加任何生长调节物质的培养基(MS、B5或SH)上只产生单苗,而在附加不同种类和不同浓度的生长调节物质的培养基上则诱导形成愈伤组织,但未能实现分化;以胡椒无菌萌发的实生苗胚轴和叶片切段作外植体进行培养,较易诱导产生愈伤组织,但难以实现分化。     

杜仲叶片和叶柄愈伤组织的诱导和植株再生

本实验以5~6年生杜仲叶片及叶柄为外植体,研究了杜仲愈伤组织诱导及植株再生的方法。结果表明:接种于补加NAA(2.0~4.0 mg/L)或BA(1.0 mg/L)+NAA(2.0~4.0mg/L)的MS培养基上的叶片和叶柄,经21~28d培养后,脱分化形成绿色或浅绿色致密愈伤组织,频率达到70%以上。绿色致密愈伤组织在补加BA(2.25~2.75 mg/L)+NAA(0.15 mg/L)的MS培养基上经过1~2次继代之后,即出现茎芽分化,频率在15%以上,只是其中许多都是畸形苗,正常苗频率较低。此问题尚在研究之中。选择生长健壮的再生植株,切除其基部愈伤组织,然后将切口浸泡在250mg/L无菌ABT生根粉溶液中3~5sec,再插入1/4强度无激素MS培养基中, 2~3周后,在苗基部长出1~3条白色粗壮的不定根,生根频率在60%以上。     

墨兰的无菌播种和植株再生

墨兰的无菌播种结果发现,在不添加细胞分裂素的培养基上,种子可以发芽,但只有原球茎和根状茎产生;不可能进一步分化成苗,只有在含有不同激素成分的MS或KnudsonC培养基上,才有可能诱导芽的分化,其中以附加6-BA 0.5-1.0mg/L+NAA 0.1mg/L诱导效果最佳,在附加6-BA 2.0mg/L+NAA 0.4mg/L的MS培养基中,能加速芽的增殖,根状茎转入含有相同激素成分的液体增殖培养基中振荡培养,可大大提高芽的分化速率。添加0.5%活性炭对芽的分化有明显增效作用。在附加NAA 0.2mg/L的MS培养基中;幼苗的生根效果最佳。     

珍稀濒危植物蒙古扁桃的组织培养及植株再生

对珍稀濒危植物蒙古扁桃进行组织培养获得再生植株。实验结果表明,在MS培养基上蒙古扁桃幼苗茎尖,茎切段和叶片等外植体均可以脱分化形成愈伤组织,并进一步分化形成再生植株。器官的脱分化与再分化决定于培养基中的激素种类及其浓度。诱导愈伤组织形成的最适培养基为MS＋6－BA0.8mg/L NAA0.1mg/L,芽分化诱导最适培养基为MS＋6－BA0.8mg/L,诱导生根的最适培养基是MS＋IBA0.5mg/L。     

Organogenesis and Plantlet Regeneration in Vitro of Populus euphratica

The research of organogenesis and in vitro plantlet regeneration of Populus euphratica Oliver was carried out using the tender shoots from mature tree as initial explants and MS medium as the basic medium. The effects of plant growth regulators (PGR) on the regeneration were compared. The results showed that the concentration of PGR was not strictly required for the organogenesis of the excised organs and callus, but the ratio of BA to NAA was important. Calli could be induced from the excised leaves and stems cultured on the medium with 0.5 mg/L BA and 0.5 mg/L NAA. The embryonic callus could be multiplied in dark on the medium supplemented with 0.25 mg/L BA and 0.5 mg/L NAA. For the adventitious bud regeneration of the leaf and callus, supplement with 0.5 mg/L BA and 0.1 mg/L NAA was appropriate, giving a regeneration frequency of 82.9% and 100%, respectively. The suitable level of BA and NAA for the excised stem's was 0.1 mg/L and 0.01 mg/L respectively, yielding a regeneration frequency of 83 %. Rooting occurred on the MS medium with half strength of macronutrient and addition of 0.015 mg/L NAA, and the rooting rate could reach up to 86.2%. The techniques of somatic cell cloning of P. euphratica was established in vitro. The problems of deterioration of the subcultured shoots were also discussed.     

Propagation of the tropical tree,Leucaena leucocephala K67, by in vitro bud culture

A tissue culture method is described for clonal multiplication of Leucaena leucocephala K67 using single lateral bud explants from 2–3 m tall greenhouse grown trees. N-6 benzyladenine (BA: 3.0 mg.1^-1) and napthaleneacetic acid (NAA: 0.05 mg.1^-1) in Murashige & Skoog's (MS) medium were found to be best suited for multiple shoot differentiation in 4–5 week old cultures. Analysis of variance of the main treatment effects of BA and NAA on shoot parameters showed that BA significantly (P=0.001) affected shoot development while NAA did not. A shoot multiplication rate of 22±3.63 shoots per bud explant was obtained in 150 days on 1/2 strength MS medium with 3.0 mg.1^-1 BA and 0.05 mg.1^-1 NAA. Shoots developed adventitious roots within 15 days in 1/2 strength MS medium containing indole-3-butyric acid (IBA: 3.0 mg.1^-1) and Kinetin (0.05 mg.1^-1). Eighty percent of the transplanted plantlets are being grown in greenhouse conditions.     

In vitro propagation of Rudraksha (Elaeocarpus sphaericus (Gaertn.) K. Schum): a biotechnological approach for conservation

The species Elaeocarpus sphaericus (Rudraksha) is a religious, medicinally important threatened tree of India. An efficient micropropagation protocol has been developed from nodal explants of this plant species collected from north-east India for large scale production of planting material at favourable sites within the country. Best shoot initiation occurred in MS medium supplemented with 2.2μM BA+2.2μM Kn in combination. Addition of Casein Hydrolysate (CH) (100mg/L) increased the shoot number. Microshoots excised and subcultured in 2.0μM BA further enhanced growth and multiplication. The shoot cultures were maintained in this concentration for 2years with subculturing at 6weeks interval. MS medium containing 5.0μM NAA was most effective for rooting. Successfully acclimatized plants (80%) showed normal growth under suitable habitat conditions.     

太子参‘柘参1号’离体快繁试验

以太子参‘柘参1号’叶片、茎段为外植体进行离体快繁试验,结果表明：带腋芽的茎段能够诱导出丛生芽,且诱导率较高,最佳诱导培养基为MS + 6-BA 1.0 mg/L + NAA 0.2 mg/L +蔗糖30 g/L;增殖培养基为MS + 6-BA 1.0 mg/L + NAA 0.2 mg/L +蔗糖30 g/L;生根培养基为MS + NAA 0.2 mg/L +蔗糖30 g/L。     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Gentiana dinarica</Emphasis> Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l⁻¹ IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.