基于响应面法的白蜡属花粉离体萌发培养基优化

基于Box-Behnken设计(Box-Behnken design,BBD)的响应面法,对绒毛白蜡(Fraxinus velutina)、新疆小叶白蜡(F.sogdiana)和美国白蜡(F.americana)3个树种花粉的离体萌发培养基成分进行了研究。以花粉萌发率为响应指标,建立了3种培养基成分(蔗糖、CaCl2和H3BO3)与花粉萌发率间的响应关系模型。在此基础上,通过无约束优化设计得到了3个树种花粉的最佳萌发条件。结果表明,蔗糖是影响花粉萌发的最主要因素,当蔗糖浓度一定时,CaCl2和H3BO3之间交互作用明显。同时还对响应面建模优化后得到的最佳萌发培养基进行了验证,结果表明:上述3种花粉的实际萌发率依次为58.33%、69.71%和59.42%,均与优化得到的理论响应值相吻合,同时也验证了基于BBD响应面模型进行花粉离体萌发条件优化方法的有效性。     

响应面法优化麻疯树花粉离体萌发培养基的研究

麻疯树因其种子含油率较高,种子油提炼的生物柴油可部分替代汽油,而成为一种极具潜能的能源作物,但由于产量低,麻疯树在热带、亚热带的发展受到极大限制。杂交育种是提高产量的重要手段,杂交亲本花粉生活力的高低直接影响到育种的成效。因此,寻求麻疯树离体花粉萌发的最适培养基配方,探明花粉萌发培养基中各主要培养基成分间的交互作用对生产上麻疯树杂交结实率和种子产量的提高具有重要意义。该研究以麻疯树开花初期雄花上花药刚散粉时的成熟花粉粒为材料,采用Box-Behnken设计(Box-Behnken design,BBD)的响应面法,对麻疯树花粉离体萌发培养基中各主要培养基成分的浓度配比及各主要培养基成分的交互作用进行了研究。以花粉萌发率为响应指标,建立了4种营养成分(蔗糖、硼酸、硝酸钙、硝酸钾)与花粉萌发率的响应面模型,并对各主要培养基成分的浓度配比进行了优化。通过R软件进行响应面分析的结果表明:4因素对花粉萌发率的影响顺序为蔗糖硼酸硝酸钙硝酸钾;蔗糖与硼酸、蔗糖与硝酸钙、蔗糖与硝酸钾之间的交互作用显著。响应面建模优化后的最佳培养基为13.77%蔗糖+32.14 mg·L~(-1)硼酸+22.21 mg·L~(-1)硝酸钙+19.95 mg·L~(-1)硝酸钾+200 mg·L~(-1)硫酸镁,在此条件下的理论萌发率为99.73%。采用此培养基成分配比得到麻疯树花粉离体试验萌发率为98.97%,与理论响应值相吻合,同时也表明利用BBD设计的响应面模型进行麻疯树花粉离体萌发培养条件优化方法的有效性。     

楸树等4种梓属树种花粉离体培养条件的研究

为筛选出楸树(Catalpa bungei C. A. Mey. )等4个树种花粉离体培养的适宜条件,以花粉萌发率和花粉管长度为指标,研究了培养时间、培养温度、液体培养基pH值、蔗糖浓度和PEG-4000浓度对2个楸树花粉样品(CB-1和CB-2)及滇楸[C. fargesii Bur. f. duclouxii (Dode) Gilmour]、黄金树[C. speciosa (Warder ex Barney)Engelmann]和梓树(C. ovata G. Don)花粉离体萌发的影响.实验结果显示,不同培养时间对楸树的花粉萌发率和花粉管长度均有极显著影响,培养至6 h时花粉萌发率最高(91.0%),培养至6～7 h时花粉管长度达到最长,最适培养时间为6 h.培养温度、液体培养基pH值、蔗糖浓度和PEG-4000浓度对4个树种花粉萌发率和花粉管长度均有明显影响.在19 ℃～36 ℃范围内,较低或较高的培养温度均对花粉萌发有一定的抑制作用,适宜各树种花粉离体萌发的培养温度为24 ℃～28 ℃;供试的4个树种花粉适宜在弱酸性环境下萌发和生长,适宜的液体培养基pH值为5.0～5.6;在蔗糖浓度为15～25 g·L-1的液体培养基中,花粉萌发率及花粉管长度均达到最大值;液体培养基中添加不同浓度PEG-4000,在较短的培养时间(3 h)内明显抑制花粉萌发和花粉管生长,但培养时间延长至6 h,添加5～25 g·L-1 PEG-4000对花粉萌发有一定促进作用,适宜的PEG-4000浓度为20 g·L-1.结果表明,不同树种甚至同一树种不同种源适宜的花粉离体培养条件有一定的差异,应根据种类或种源进行适当的调整.     

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。     

紫花含笑（♀）、灰岩含笑（♂）及其杂种F1代花粉萌发试验研究

对紫花含笑(Michelia crassipes)、灰岩含笑(M.calcicola)及其杂种F1代花粉生活力进行了研究,为基于紫花含笑和灰岩含笑杂种F1代的含笑属观赏植物新品种培育与种质创新提供科学数据及研究资料.研究发现,亲本(紫花含笑和灰岩含笑)新鲜花粉萌发率均可达90％以上,杂种F1代花粉萌发率从38％到79％不等,平均为57.7％,低于双亲.亲本及其杂种F1代花粉萌发的最适温度为25℃,温度过高花粉管的伸长受到抑制,并导致花粉管顶端破裂.亲本及多数杂种F1代的新鲜花粉在100 g/L和150 g/L的蔗糖浓度下萌发率都较高;经-20℃贮藏后的花粉对蔗糖浓度的敏感性要高于新鲜花粉.杂种F1代及其亲本的花粉在离体培养中均会出现双萌发管现象.番红染料对液体培养基中的花粉有致死和染色作用,有利于统计杂种F1代及其亲本的花粉萌发率.     

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%。     

紫斑牡丹单瓣与半重瓣花粉超微结构及生理生化特性比较北大核心CSCD

该研究以3个紫斑牡丹品种(‘象牙白’、‘美人面’、‘紫金冠’)的单瓣与半重瓣花粉为材料,对其超微结构和生理生化特性进行观察分析,并对其花粉培养基进行筛选优化,探讨单瓣和半重瓣花粉的超微结构与花粉萌发率以及物质代谢与花粉萌发率之间的关系。结果表明:(1)紫斑牡丹花粉萌发的最适宜培养基为100 g·L^(-1)蔗糖+0.15 g·L^(-1)硼酸+10 g·L^(-1)琼脂。(2)花粉饱满率是影响花粉萌发的主要原因。(3)3个紫斑牡丹品种的单瓣花粉离体培养萌发率均显著高于半重瓣的花粉。(4)‘象牙白’、‘美人面’、‘紫金冠’单瓣花粉可溶性蛋白含量均高于半重瓣花粉,但三者单瓣花粉的MDA含量和3种保护酶(CAT、SOD、POD)活性均低于半重瓣花粉。研究认为,花粉体内蛋白质亏损和MDA、CAT、SOD、POD的积累可能是引起半重瓣紫斑牡丹花粉萌发率较低的生理原因之一。     

梓树属花粉生活力的研究

以梓树属楸树组3种楸树花粉为试材,采用固体培养基法进行离体培养,研究不同浓度蔗糖、硼酸对花粉萌发的影响,以确定最适宜培养基条件下最佳的培养时间.结果表明,3个种花粉的最佳离体萌发条件一致.培养基中含有20%的蔗糖、0.005%的硼酸时较适合3种楸树花粉萌发,楸树、灰楸和滇楸萌发率分别为28.34%、12.24%和60.22%;3个种的花粉最佳培养时间均为6 h;不同重复数之间萌发率差异不显著,以3个重复为宜.花粉种内单株之间萌发率差异显著,且滇楸花粉萌发率明显高于楸树和灰楸.     

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%.     

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。     

Germination and Storage of Pollen of Phytolacca dodecandra L. (endod)

The effect of sucrose, H₂BO₃, KNO₃, Ca(NO₂)₂.4H₂O and MgSO₄.7H₂O on pollen germination of Phytolacca dodecandra L. (endod)in a liquid medium was investigated. Sucrose and H₃BO₃ werecritical to pollen germination. A concentration of 10% sucroseand 161.8 µm H₂BO₃ gave over 70% germination. The germinationof pollen was not enhanced by Ca(NO₃)₂.4H₂O, KNO₃ and MgSO₄.7H₂O.Endod pollen was dehydrated over CaCl₂ and stored in gelatincapsules in cryogenic vials at –175 °C, 1±1°C and 24±2 °C. The pollen moisture content atcollection was approx. 7.8% (f. wt basis) and dehydration overCaCl₂ reduced it to about 1.4%. Pollen stored at 1±1°C and –175 °C maintained viability for over 6months. Pollen stored at room temperature lost viability within4 weeks of storage. Pollination with cryopreserved pollen resultedin normal fruit set. Phytolacca dodecandra, endod, pollen germination, pollen storage     

景宁木兰花粉萌发与贮藏特性研究

以景宁木兰的花粉为试材,采用花粉离体培养法,用单因子与正交试验研究不同浓度蔗糖、H3BO3、CaCl2所组成的基本培养基对景宁木兰花粉萌发的影响,同时探讨不同贮藏条件和贮藏时间对花粉活力的影响。结果表明:景宁木兰在30 g·L-1蔗糖+200 mg·L-1H3BO3+200 mg·L-1CaCl2的液体培养基上萌发率最高(74.56%)。低温条件下有利于景宁木兰花粉生活力的保持,在-80℃条件下,花粉生活力下降较慢,并且随着贮藏时间的增加,经过硅胶干燥的花粉的活力明显高于湿润花粉的活力。     

In vitro chemotropism of pearl millet pollen tubes to stigma tissue: a response to glucose produced in the medium by tissue-bound invertase

Summary Water-homogenized stigma pellets of pearl millet and precipitates resulting from dialysis of their salt extracts were observed to: (1) chemotropically attract pearl millet pollen tubes on a sucrose-containing pollen germination and growth medium, (2) have acid invertase activity as assayed by the arsenomolybdate method, (3) hydrolyze sucrose in the pollen germination and growth medium to glucose as assayed by coupled glucose oxidation with Nitro Blue Tetrazolium, and (4) lose chemotropic and invertase activities upon heat treatment. The results indicate that the in vitro chemotropic attraction of pearl millet pollen tubes to water-homogenized stigma pellets is a response to glucose produced by homogenate-pellet-bound invertase hydrolyzing the sucrose present in the pollen germination and growth medium. Yeast and tomato invertases used as controls verified this conclusion. Water extracts of whole stigmas contained water-soluble acid invertase. The results are discussed in relation to the identification of possible in vivo chemotropic factors of pearl millet and other plants by in vitro assays.Abbreviations dH₂O Deionized, house-distilled water - NBT Nitro Blue Tetrazolium, NBT-medium - PGG medium, pollen germination and growth medium (10% sucrose, 1 mM H₃BO₃, and 1% agarose); - WHS pellet, water-homogenized stigma pellet On Specific Cooperative Agreement 58-6612-8-002 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

A rapid,efficient method for the mass production of pollen protoplasts from Pinus bungeana Zucc. ex Endl. and Picea wilsonii Mast.

An optimized protocol was established to isolate large numbers of mature living pollen protoplasts of Pinus bungeana Zucc. ex Endl. and Picea wilsonii Mast. Intact pollen grains of P. bungeana or pollen with short tubes were incubated with gentle agitation in a solution of 2% cellulase R-10, 1.5% macerozyme R-10, 15% sucrose, 0.01% H₃BO₃, and 0.01% CaCl₂. Intact pollen protoplasts with diameters of 40 μm were liberated, with an isolation rate of up to 70% after 6 h of enzymatic incubation. The optimal pH and temperature for the reaction were 5.8 and 24 °C, respectively, and the optimal enzymatic digestion conditions were 6 h of incubation in the above solution. The method for isolating pollen protoplasts from P. wilsonii was similar to that for P. bungeana, except that the incubation medium contained 12% rather than 15% sucrose and the optimal enzyme concentrations were 3% cellulase and 2% macerozyme. The isolated pollen protoplasts were demonstrated to be living by microscopy in a fluorochromatic reaction with fluorescein diacetate (FDA).     

Evaluation of Viability,Shedding Pattern,and Longevity of Pollen from Genetically Modified (GM) Herbicide-tolerant and Wild-type Zoysiagrass (<Emphasis Type="Italic">Zoysia japonica</Emphasis> Steud.)

Knowledge about pollen viability is important when evaluating the risk of genetically modified (GM) plants. Here, staining via iodine potassium iodide (IKI) or triphenyl tetrazolium chloride (TTC) could not distinguish between live and dead pollen from Zoysia japonica. Therefore, to obtain a reliable assessment of such viability and longevity, we developed an optimum germination medium containing 20% sucrose and 50 ppm H₃BO₃. Pollen grains transferred to the germination medium at about 1000 hours had a germination rate of >90%. Pollen was most predominantly shed at approximately 1000 hours, with viability declining to nearly 0% at 1200 hours. All germinability was lost within 150 min when stored at 25°C. No significant difference was found between GM and non-GM plants in their pollen viability or longevity.     

The in vitro germination and storage characteristics of Keteleeria fortunei var. cyclolepis pollen provide a reference for cross breeding

Keteleeria fortunei var. cyclolepis is an ideal tree species for mountain afforestation, timber forests, and landscaping. Its pollination process can be affected by the rainy season, making it difficult to pollinate the massive female cones, which leads to a high abortion rate and low quality of seeds. Here, we observed the pollen morphology of K. f. cyclolepis using scanning electron and light microscopes, investigated the characteristics of its in vitro germination by the detached method, and explored the effect of different storage temperatures and times on the pollen germination rate and the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Our results indicated that the pollen of K. f. cyclolepis is a five-cell pollen, comprising one noumenon and two air sacs, both of which were oval in polar view. The optimal condition for pollen germination of K. f. cyclolepis was 240 g/L sucrose + 70 mg/L CaCl₂ + 210 mg/L H₃BO₃ at 24 °C and pH 6.0, resulting in a germination rate of 45.0%. The effects of different storage temperature and time on pollen germination rate varied significantly. The best storage temperature was − 80 °C, at which the germination rate was 20.9% after 365 days of storage, and the activity of three protective enzymes remained relatively high, representing relatively strong antioxidation and antiaging activity. Stepwise regression analysis showed that SOD was the main factor affecting the pollen germination rate of K. f. cyclolepis. The function of the three protective enzymes differed under various temperatures, for example, SOD served as a sensitive protective enzyme at room temperature, − 20 °C and − 80 °C, whereas both SOD and CAT served as sensitive protective enzymes at 4 °C.

     

Effects of nutrients present in Bold’s basal medium on the green algaStigeoclonium pascheri

The effects of varying concentrations of nutrients present in Bold’s basal medium on the extent of colony formation from vegetative fragments, sporulation and spore germination of the green algaStigeoclonium poscheri were studied. A decrease of colony formation was observed in media deficient in MgSO₄, NaNO₃, phosphates, and containing a 10-fold increase of H₃BO₃. Sporulation decreased in the same media. However, sporulation was greater in an increasing order in media containing 2- to 10-fold increase in MgSO₄. There was a decrease in spore germination in media deficient in phosphates, MgSO₄, containing 5- or 10-fold MgSO₄, or containing 2- to 10-fold of CaCl₂, H₃BO₃ or microelements. Spore germination increased in media containing 2-fold MgSO₄, deficient in H₃BO₃ or microelements or containing none of the three micronutrient solutions.     

EFFECT OF SALINITY ON POLLEN I. POLLEN VIABILITY AS ALTERED BY INCREASING OSMOTIC PRESSURE WITH NaCl,MgCl2, and CaCl2

Petunia (Petunia hybrida Vilm. cv. ‘Snowstorm') plants were grown in saline solution (NaCl, MgCl₂, and/or CaCl₂) of 0, 1, 2, and 3 bars osmotic pressures. Pollen viability was tested by tetrazolium chloride staining and by germination (by the hanging drop method, using 15 % sucrose and 0.01 % boric acid as the nutrient medium, at 27 ± 1 C). Pollen viability decreased with increased salinity. Pollen from plants grown in single salt solutions of NaCl, MgCl₂, and CaCl₂ (each at 0, 1, 2, or 3 bars osmotic pressure) was germinated in base culture medium. Pollen viability decreased more with NaCl than with MgCl₂ or CaCl₂. In vitro studies of the effects of three salts, viz., NaCl, MgCl₂, and CaCl₂, on pollen germination and tube growth showed that NaCl inhibited germination and pollen tube growth more than did MgCl₂ or CaCl₂. MgCl₂ was least injurious, and even promoted tube growth at 0.5 and 0.75 bars osmotic pressure. Adding low concentrations of MgCl₂ reduced the toxic effect of NaCl and increased the percentage of germination. CaCl₂ reduced the effect of NaCl less than did MgCl₂. We conclude that specific ion effects were more important than osmotic pressure.     

Use of Vital Dyes in Conjunction with the Semivitro Technic for the Detection of Pollen Tube Sperm Nuclei

The technique we describe here is a modification of that used by Hough et al. (1985), combined with “semivitro” pollen tube observations. With the semivitro technique, pollen tubes grow from the cut ends of pollinated styles (Brewbaker and Majumder 1961). Pollen of Nicotiana alata was presoaked for 15 min in simplified medium (Brewbaker and Kwack 1963) (10% sucrose, 300 ppm Ca(NO₃)₂, 100 ppm H₃BO₃ with the addition of 0.5 mg/ml of Hoechst 33258 stain from Serva Biochemicals, Heidelberg, Control H, purchased June 1983). (For germination of Nicotiana alata pollen in vitro, we use this same solution, except with 12% sucrose). After this prestaining, the pollen suspension was centrifuged for 5 min at 1200 × g, the pellet resuspended in control Brewbaker medium (i.e., no stain), recentrifuged and used to pollinate detached pistils. The pistils were then incubated at 25 C in a water-saturated atmosphere for 20 hr. At this time, the styles were cut just ahead of the front of the growing pollen tubes (Mulcahy and Mulcahy 1985) and the cut stylar ends each dipped in fresh control Brewbaker medium. Twelve to 24 hours later, tubes growing out of the cut styles were viewed by fluorescence microscopy (exciter filter, BG 12 + KV 418, beam splitter, 500 nm, and barrier filter OG 515). A distinct green fluorescence was seen in the generative and vegetative nuclei (Fig. 1).