Germination of trinucleated pollen: formulation of a new medium for Capsella bursa-pastoris

Summary In Brewbaker and Kwack's medium (BK) only 16% of the pollen grains germinated, and these produced pollen tubes having a maximum length of 25 m. With a solution based on Monnier's medium 47% germination and 160-mlong pollen tubes were observed. Calcium was shown to be essential for germination; the optimal concentration was 880 mg/l calcium chloride. The optimal concentrations of magnesium sulphate and boric acid were 360 and 50 mg/l, respectively. Germination at pH 4.0 but also pH 8.0 and the presence of vitamins B1 and B6 (1 mg/l each) were stimulatory. Polyethylene glycol (PEG) was superior to sucrose as an osmoticum and germination and tube length were significantly improved using PEG 4000 at a concentration of 120 g/l (0.03 M). Equimolar concentrations of PEG 400 and PEG 600 gave inferior results. Combining PEG with sucrose in the medium did not improve germination or increase tube length.     

培养基组分及培养条件对蜡梅花粉萌发及花粉管生长的影响

采用液体培养法研究不同培养基组分和培养条件对蜡梅花粉萌发和花粉管生长的影响。结果表明:(1)PEG-4000是蜡梅花粉离体培养所必需的培养基成分,当培养基中无PEG-4000时,花粉不能正常萌发。(2)培养基内低浓度蔗糖对花粉萌发和花粉管的生长无显著影响,但随着蔗糖浓度的升高,则对花粉萌发和花粉管生长表现出强烈的抑制作用,且浓度越高,抑制效应越强。(3)培养基内其它组分分别在一定浓度范围(0～250g/L PEG-4000、0～50mg/L硼酸、0～30mg/L硝酸钙)内对花粉萌发及花粉管生长有促进作用,但超过上述高限值时则起抑制作用。(4)培养基内镁和钾的浓度对花粉萌发及花粉管生长影响不显著。研究表明,蜡梅最适花粉液体培养基组分为250g/L PEG-4000+50mg/L H3BO3+30mg/L Ca(NO3)2.4H2O,且在pH 5.5、温度15℃和600lx的光照培养条件下蜡梅花粉萌发和花粉管生长最佳。     

梅花花粉离体萌发和花粉管生长研究

(南京农业大学园艺学院,南京210095)摘要:研究培养基成分、pH值和培养方式对梅花花粉离体萌发和花粉管生长的影响。结果表明:不同品种梅花花粉离体萌发的最适培养基为ME3+200g.L-1PEG4000(pH5.0),品种‘淡丰后’、‘久观绿萼’、‘喧妍宫粉’和‘月光玉蝶’最高萌发率可分别达到58.6%、60.6%、85.6%和50.7%。PEG4000能显著促进梅花花粉萌发,在培养基各成分中作用最大,不可替代。低浓度(50g.L-1)蔗糖对梅花品种花粉萌发作用不显著,而高浓度(≥100g.L-1)蔗糖明显抑制花粉萌发和花粉管生长。固体和液体培养对梅花花粉离体萌发的影响差异不显著。     

培养条件及贮藏温度和时间对木麻黄花粉萌发率的影响

用离体培养的方法研究了不同蔗糖、硼酸浓度,以及不同贮藏温度和贮藏时间对木麻黄(Casuarina)花粉萌发的影响.结果表明:15%的蔗糖是木麻黄花粉萌发的最佳浓度;在15%蔗糖培养基上添加硼酸显著促进木麻黄花粉萌发,250 mg kg-1硼酸是木麻黄花粉萌发的最佳浓度;添加了琼脂的固体培养基更有利于木麻黄花粉萌发;在常温下木麻黄生活力丧失很快,但低温下花粉的萌发力可保持较长时间.     

Reduction of vitrification in in vitro raised shoots of Chlorophytum borivilianum Sant. & Fernand., a rare potent medicinal herb

Reduction of vitrification in in vitro raised shoots derived from shoot bases and immature floral buds along with inflorescence axis used as explants of C. borivilianum, a rare medicinal herb is described. Shoot multiplication was obtained on MS medium with 2 mg l(-1) benzylaminopurine (BAP) + 0.1 mg l(-1) indole-3-butyric acid (IBA) and MS medium with 2 mg l(-1) kinetin (Kin) + 0.1 mg l(-1) 2,4-dichlorophenoxy acetic acid (2,4-D) from shoot bases and inflorescence axis respectively. Best multiplication rates were obtained from both the explants on MS medium with 2 mg l(-1) BAP. Vitrification of shoots in cultures appeared during the multiplication stage. Culture bottles with aerated caps reduced the vitrification to 80%. Reduction of BAP concentration from 2 mg l(-1) to zero during subsequent subcultures also minimized vitrification. Use of 0.5-2 mg l(-1) Kin produced healthy shoots when compared to BAP. In vitro raised shoots rooted on Knop salts containing iron and vitamins of MS medium, 2 mg l(-1) IBA and 0.1% activated charcoal. About 80% plantlets survived upon soil transfer. Scanning electron microscopic and image analyzer studies reveal the morphological structural differences between the leaves of normal and vitrified plantlets.     

甘蓝型油菜花粉离体培养

通过对甘蓝型油菜花粉发育阶段和活力的检测确定花粉发育的时期,分离出单核晚期花粉进行离体培养.结果表明,(1)筛选出适合油菜小孢子花粉离体培养的液体培养基为T_1+怀特维生素(White's vitamins)+2%椰子汁+0.5 mol/L麦芽糖,在此培养基上花粉的成熟率可达25.1%,萌发率达6.3%.(2)筛选出适合成熟花粉离体萌发液体培养基为0.6 mol/L麦芽糖+1.6 mmol/L硼酸+2.9 mmol/L硝酸钙+29.6 μmol/L VB_1,在此培养基上,自然成熟花粉的萌发率可达75.2%.将离体培养成熟的花粉培养在萌发培养基,萌发的花粉占成熟花粉的66.3%.     

In vitro clonal propagation of Chlorophytum borivilianum Sant. et Fernand., a rare medicinal herb from immature floral buds along with inflorescence axis

A novel method of shoot regeneration from immature floral buds along with inflorescence axis in C. borivilianum, a rare medicinal herb is described. Using this explant, axenic cultures were established with very less contamination (10%). MS medium with 2 mg l(-1) kinetin and 0.1 mg l(-1) 2, 4-dichlorophenoxyacetic acid proved to be the best for multiple shoot induction. Maximum number (35) of shoot production was achieved in MS medium with 2 mg l(-1) benzylaminopurine. Rooting of shoots (86.7%) with maximum fasciculated roots (5) occurred on Knops medium containing iron and vitamins of MS medium with 2 mg l(-1) indole-3-butyric acid and 0.1% activated charcoal. Plant survival was 80% in four weeks after their removal from in vitro conditions. Per explant 34 hardened plants generated within 50 weeks. This protocol can be useful for large-scale clonal multiplication from immature floral buds with inflorescence axis and successfully used for germplasm conservation of this rare medicinal herb without destroying the mother plant.     

In vitro propagation of shoot buds of Carica papaya L. (caricaceae) var. Honey Dew

Shoot buds from the saplings and the fruit bearing plants of Carica papaya L.. var. Honey Dew (papaya) initially treated with Gentamycin were cultured in modified MS media, each with a different hormonal combination, for the establishment of cultures and multiplication and rooting of plants. About 43% of explants from fruit bearing plants and 69% of those from saplings remained free of contamination and retained regeneration capacity when treated in 500 mg/l Gentamycin. For the establishment of the explants a medium containing 1 mg/l GA₃ and 2 mg/l kinetin was necessary. When established buds were transferred to medium containing 1 mg/l NAA and 3 mg/l kinetin, calli were initiated at cut ends of shoot buds; multiplication started on transfer to NAA (0.1 mg/l) and BAP (0.5 mg/l) medium. Cultures have been maintained for the last twenty months without any loss in multiplication rate. Rooting was induced in medium with reduced salt concentration containing 2 mg/l IBA. Shoot elongation was induced after prolonged culture in the same rooting medium.Abbreviations MS Murashige and Skoog, 1962 - SH Schenk and Hildebrandt, 1972 - GA₃ Gibberellic acid - Kn Kinetin - NAA Napthaleneacetic acid - BAP 6 -Benzylaminopurine - IBA Indole-3-butyric acid - IAA Indole-3-acetic acid     

Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Helianthus annuus L.

We have developed a highly efficient three-stage protocol for plant regeneration in sunflower (Helianthus annuus L.) from embryonal cotyledons. This protocol uses phenylacetic acid (PAA) for both shoot-bud induction and the elongation of smaller buds. The medium used for inducing bud formation from the cotyledons was modified MS medium supplemented with 3 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l PAA. Buds were elongated on MS medium supplemented either with only 0.2 mg/l gibberellic acid (GA₃) or with 0.2 mg/l GA₃ + 0.1 mg/l PAA + 0.3 mg/l BAP. The elongated shoots were then transferred onto rooting medium containing 1 mg/l PAA. The complete plantlets with well-developed roots were transferred to field conditions where they survived and set normal seeds. The induction of shoot buds from embryonal cotyledons was also observed on modified MS medium supplemented with 0.5-5 mg/l BAP in combination with 0.5-5 mg/l !-naphthaleneacetic acid (NAA). In this case, the formation of callus took place along with shoot-bud formation, which hindered further development of the latter. The presence of PAA with BAP in the primary bud induction medium promoted normal development and elongation of shoot buds.     

菜心组织培养技术初探

为建立菜心(Brassica campestris ssp.chinensis var.utilis)的快繁技术体系,以花药和子叶-子叶柄为外植体进行组织培养研究。结果表明,花药培养以选取未开放的花蕾为宜,且花柱略高于花瓣,此时小孢子多数处于单核靠边期。菜心花粉的萌发率不高,且秋冬季的花粉比夏季的萌发率高。菜心花药愈伤组织诱导培养基为:MS+1.0 mg L–1 KT+1.0 mg L–1 2,4-D+3%糖+6 g L–1琼脂+8%椰乳,不定芽诱导培养基为:MS+2.0 mg L–1 6-BA+0.5 mg L–1 NAA+1.0 g L–1活性炭+2%糖+6 g L–1琼脂或MS+2.0 mg L–1 ZT+0.5 mg L–1 IAA+0.5 g L–1 AgNO3+1.0 g L–1活性炭+2%糖+6 g L–1琼脂。花药培养的不定芽诱导率为36.7%,不定芽培养出现褐化现象,不能形成再生植株;而以子叶-子叶柄为外植体培养获得的植株再生率可达80%。     

温度、蔗糖和硼酸对含笑花粉离体萌发的影响

以含笑(Michelia figo)花粉为试验材料,采用花粉人工培养法研究了含笑花粉离体萌发的最适条件。结果表明:蔗糖、硼酸在一定浓度范围内能促进花粉萌发,含笑花粉萌发的最适蔗糖和硼酸分别为50 mg.L-1和5mg.L-1,萌发率分别为21.51%和44.14%;花粉最佳萌发温度为25℃,萌发率达53.33%。正交试验设计结果表明,花粉萌发的最佳组合为70 mg.L-1蔗糖+5 mg.L-1硼酸+28℃,萌发率可达65.33%。     

Staining for viability testing,germination and maturation of Sambucus nigra L. pollen in vitro

Freshly released pollen of black elderberry (Sambucus nigra L.) was incubated under various culture conditions until germination was achieved. Optimal conditions for germination were determined and used for maturation of unicellular microspores in vitro. Staining with 5-diphenyltetrazolium bromide, propidium iodide and iodine potassium iodide was used to assess pollen viability, nuclear phase and maturation, respectively. The germination rate was highest when fresh pollen was agitated at 40 rpm in Petri dishes containing a liquid medium consisting of Brewbaker and Kwack salts, 15% (w/v) sucrose, 500 mg/l MES sodium salt, at pH 5.0; germination reached nearly 70% after only 1 h in culture. Under these conditions, and with addition of 200 mg/l glutamine, 260 mg/l cytidine and 500 mg/l uridine, uninucleate microspores developed into mature pollen at a 12% germination rate. Our report is the first demonstration of maturation of S. nigra microspores in vitro.     

In vitro microrhizome production in Zingiber officinale Rosc.

Microrhizomes of Zingiber officinale were successfully produced from tissue culture derived shoots by transferring them to liquid MS medium supplemented with 1 mg/l BAP, 2 mg/l calcium pantothenate, 0.2 mg/l GA₃ and 0.05 mg/l NAA for shoot proliferation. After 4 weeks of incubation, the medium was replaced with microrhizome induction medium, consisting of MS salts supplemented with 8 mg/l BAP and 75 g/l sucrose. Microrhizome formation started after 20 d of incubation in stationary cultures at 25+1 ° in the dark. Microrhizomes with 1–4 buds and weighing 73.8 to 459 mg each were harvested after 50–60 d. After storage for 2 months in moist sand at room temperature, 80% of the microrhizomes sprouted producing roots and shoots.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

Somatic embryogenesis and plant regeneration from leaf callus of Ocimum basilicum L

A effective protocol for complete plant regeneration via somatic embryogenesis has been developed for Ocimum basilicum L. Callus was initiated from leaf explant of young plant on supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) 1.0 mg l(-1), 3% sucrose and 0.9% agar. The calli showed differentiation of globular structure embryos when transferred to MS medium containing 2,4-D 0.5 mg l(-1) and BAP 1.0 mg l(-1). The maximum globular structure embryos were further enlarged and produced somatic embryos in MS basal medium supplemented with BAP 1.0 mg l(-1)+NAA 1.0 mg l(-1) + KN 0.5 mg l(-1). Continued formation of globular embryo and germination of embryos occurred in this medium. Complete plantlets were transferred onto specially made plastic cup containing soilrite followed by their transfer to the garden soil. Survival rate of the plantlets under ex vitro condition was 80%.     

东方百合花粉萌发培养基组分的优化

以东方百合6个品种的花粉作实验材料,选用22个培养基配方,在研究硼酸和蔗糖浓度对花粉萌发和花粉管伸长影响的基础上,对东方百合花粉培养基配方进行了优化。研究结果表明东方百合离体花粉萌发培养基的最佳组成是:蔗糖13%,硼酸143mg·kg^-1,琼脂1%。     

Callus formation and plant regeneration through direct culture of isolated pollen of Hordeum vulgare cv. ‘Sabarlis’

Summary Pollen calli and plantlets of Hordeum vulgare cv. Sabarlis were obtained through direct pollen culture without pretreatment of spikes or preculture of anthers. Isolated immature pollen grains were cultured first in a 0.3 M mannitol solution or a C1 basal medium (Chen et al. 1979) supplemented with 0.3 M mannitol but without sucrose for 5–7 days, then transferred into a C1 medium containing 6% sucrose, 3 mM glutamine and 5 mM m-inositol. After a 3 week culture period small pollen calli derived from the pollen grains were transferred into a growth medium comprising C1 basal medium supplemented with 250 mg/1 lactalbumin hydrolysate and 0.5 mg/1 kinetin. For shoot regeneration, vigorously growing calli were transferred onto agarsolidified MS medium (Murashige and Skoog 1962) containing 3% sucrose, 2 mg/1 benzyladenine and 0.5 mg/1 indole-3-acetic acid. The ratio of green plants to albino was approximately 12.2.     

In vitro plant regeneration of fig (Ficus carica L. cv. gular) using apical buds from mature trees

A reliable procedure for multiple-shoot induction and plantlet regeneration was developed with apical buds collected from 7- to 8-year-old trees of Ficus carica L. using Murashige and Skoog's (MS) medium supplemented with 2.0 mg/l 6-benzylaminopurine and 0.2 mg/l 1-naphthaleneacetic acid. The in-vitro-regenerated shoots were further multiplied on MS medium supplemented with 2.0 mg/l 6-benzylaminopurine and 0.2 mg/l 1-naphthaleneacetic acid and an average multiplication rate of four per subculture was established with 90% success. Excised shoots were rooted in liquid half strength MS medium supplemented with 2.0 mg/l indole-3-butyric acid and 0.2% activated charcoal. Regenerated plantlets were successfully established in soil, with a success rate of 68%. Received: 28 July 1997 / Revision received: 15 January 1998 / Accepted: 7 February 1998     

Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Capsicum annuum L.

 A highly efficient three-stage protocol for the regeneration of chilli pepper (Capsicum annuum L.) from cotyledon explants was developed. This protocol used PAA in both the shoot-bud induction medium and the medium for elongation of the shoot buds. A superior medium for the induction of buds from the cotyledons was MS medium supplemented with BA (5 or 7 mg/l) + PAA (2 mg/l). Buds were elongated during the second stage on medium containing BA (2 or 5 mg/l) + PAA (2 mg/l). On this medium most of the buds elongated, and their number also increased due to the formation of new buds; bud elongation was achieved in 100% of the cultures provided the buds were induced in the primary stage on a medium supplemented with BA+PAA. The shoots that elongated in the second-stage rooted at 100% frequency on a medium supplemented with NAA (1 mg/l). The complete plantlets with well-developed root and shoot systems were transferred to field conditions where they grew to maturity, flowered and fruited normally. While shoot-bud induction from the cultured cotyledons was also observed on media supplemented with BA (5 or 7 mg/l) alone or in combination with IAA (0.2–2 mg/l), buds induced on these media were often distorted, with most not developing into normal shoots in the second-stage subculturing; a rosette of buds was seen in the second stage subculturing. On the other hand, PAA in combination with BA in the primary induction medium and second-stage medium promoted normal development and the elongation of shoot buds. Received: 28 July 1998 / Revision received: 22 December 1998 / Accepted: 19 February 1999     

杂种卡特兰花粉萌发和花粉贮藏性研究

以杂种卡特兰品种‘绿世界’(Rhyncholaeliocattleya Sung Ya Green‘Green World’)的新鲜花粉为试材,采用正交试验比较了蔗糖、H3BO3、CaCl2对花粉萌发的影响。结果表明,适宜的花粉培养基为:蔗糖100 gL-1+H3BO3 40 mgL-1+CaCl2 150 mgL-1。不同贮藏方法对杂种卡特兰花粉萌发率的影响较大,4℃低温湿润贮藏30 d后,花粉萌发率仅为3.5%,而4℃低温干燥贮藏、-20℃冷冻湿润和干燥贮藏720 d后,花粉仍然具有较高的活力,花粉萌发率均达32.5%以上,且对授粉结实率没有明显影响。杂种卡特兰的授粉最佳时期为开花后0-8 d,花粉块在整个花期均保持较高的活性。     

苏铁属花粉萌发及保存条件研究

以不同浓度梯度的蔗糖与硼酸组合在不同pH条件下用悬浮培养法测定德保苏铁、叉叶苏铁、元江苏铁和越南篦齿苏铁花粉的活力;将元江苏铁和越南篦齿苏铁花粉分别保存在不同低温、不同湿度的环境中,研究温度和湿度对保存花粉的影响。结果表明:(1)最适合苏铁属植物花粉萌发的培养液配方为蔗糖(1%～2.5%)+硼酸(100～500 mg/L),pH6.0～7.0;(2)在室温下,将苏铁花粉密封保存在有干燥剂的容器中,可存活30 d以上;(3)在0℃条件下,不加干燥剂,花粉可保存4个月以上;(4)用液氮保存后的越南篦齿苏铁花粉进行人工授粉,结实率高达90.3%,与用新鲜花粉人工授粉的结实率无明显差异;(5)将花粉含水率降低到15.5%～13.2%后,能在液氮中进行长期保存,表明花粉液氮保存可以作为苏铁花粉长期和超长期保存的方法。