Isolation of Pollen Protoplasts in Nicotiana tabacum

A new method for isolation of quantities of mature pollen protoplasts in Nicotiana tabacum has been established. The first step was to germinate mature pollen in Brewbaker and Kwack medium containing 20% sucrose. When most of the pollen grains had just germinated short pollen tubes, they were transferred to an enzymatic solution for the second step. The enzymatic solution contained 1% pectinase, 1% cellulase, 0.5% potassium dextran sulfate, 1 mol/L mannitol, 0.4 mol/L sorbitol in Dx medium with or without 15% Ficoll. The enzymes firstly degraded the pollen tube wall and then the intine. As a result, intact pollen protoplasts were released with the isolation rate up to 50%-70%. Factors affecting pollen protoplast isolation during the germination and maceration of pollen grains were studied. The suceees depended on two key points:pollen germination duration and osmotieum concentration. The optimal germination duration was 30 rain at 30℃. When it was too long, long pollen tubes formed and subsequently, large number of subprotoplasts instead of whole protoplasts were yielded, as the case reported by previous investigators. The optimal concentration of mannitol and sorbitol in enzyme solution was as high as 1.4 mol/L in total. Lowering of the osmoticum concentration resulted in decrease of percentage of pollen protoplasts.     

Regeneration of Hybrid Plantlets Via Pollen-Hypocotyl Protoplast Fusion in Brassica spp.

It has been reported that "gameto-somatic hybridization" was induced by fusion of microspore tetrad protoplasts with somatic protoplasts in Nicotiana and Petunia. However, since the success of isolation of pollen protoplasts in recent years, the use of protoplasts at pollen stage as one of the fusion partners in such hybridization is a novel experimentation. Young pollen protoplasts were isolated from the pollen grains of Brassica chinensis at mid-late unicellular to early bicellular stage the pollens for 1.5--2.5 h at 25℃ in a CPW solution containing 0.8 % of eellulase, 0.5 % pectinase, 0.1% pectolyase, 1 3 % mannitol, 1 0 % glucose, 0. 3% potassium dextran sulphate and 3 mmol/L MES. The purified pollen protoplasts were then fused with the hypocotyl protoplasts of B. napus by PEG method. Heterokaryons were identified by means of visualization of the fluorescence from FITC-prela-beled pollen protoplasts. In order to increase heterokaryons and reduce hypocotyls homokaryons, the denstity of hypocotyl protoplasts were lowered and the ratio of the number of hypocotyl vs. pollen protoplasts were adjusted from 1 : 3 to 1 : 6. The fusion products were cultured in a liquid KM8p medium supplemented with 0.4 mol/L glucose, 0.8 mg/L 2, 4-D, 0.25 mg/L NAA. 0. 5 mg/L BA, 500 mg/L glutamine and 3 mmol/L MES where cell division and callus formation took place. The calli, after being transferred to a MS medium supplemented with 2.0 mg/L BA, 3 % sucrose and 0.4 % agarose, differentiated into a few shoots. The shoots were transferred onto a half-strength MS medium supplemented with 2% sucrose, 0.1--0. 2 mg/L NAA, 0.5 mg/L IBA and 20% potato juice for root formation. Finally, three plantlets were regenerated. Chromosome counts by roottip squash method revealed that one plantlet was 2n= 48, corresponding to an allotriploid resulted from a fusion between one pollen protoplast of B. chinensis (2n = 20) and one hypocotyl protoplast of B. napus (2n = 38), and the other two plantlets were 2n = 58, which might be an allotetraploid originated from a fusion between two pollen protoplasts and one hypocotyl protoplast. The isozyme patterns of leaf esterases showed that all the three plantlets had bands characteristic of both parents. This is the first case of success in "gameto-somatic hybridization" by using pollen protoplasts rather than tetrad protoplasts as the haploid partner.     

Fluorescence Microscopic Observations on Actin Filament Distribution in Corn Pollen and Gladiolus Pollen Protoplasts

Actin filament (AF) distribution in Zea mays pollen and Gladiolus gandavensis pollen protoplasts was localized by FITC conjugated phalloidin fluorescence microprobe. The pollen was incubated in Brewbaker and Kwack (BK) medium, and the pollen protoplasts were isolated enzymatically and cultured in K3 medium containing various supplements by a previously reported method. Samples were fixed for 30 min with 1.5% paraformaldehyde dissolved in 0.1 mol/1 phosphate buffer (pH 7), half strength of BK elements, 1 mol/1 EGTA and sucrose, stained for 30–60 min with 1 μg/ml FITC-phalloidin in the buffer solution, and observed by a fluorescence microscopy. In hydrated corn pollen grains, the AFs constituted an irregular network. Prior to germination a part of the pollen grains showed polarized pattern of Afs. At the opposite pole to the germ pore, there was a center from which AF bundles radiated and converged toward the pore, often making a spindle-shaped configuration. In just isolated gladiolus pollen protoplasts, the AFs appeared as irregular fine network. After 4–7h of culture, the AF distribution coincided in some cases with the unevenly regenerated new wall area as exhibited by FITC-phalloidin and Calcofluor White ST double staining, indicating a possible involvement of AF in wall synthesis. After 17–18 h of culture, a part of the pollen protoplasts went on germination. The AFs became polarized in such protoplasts and converged into the tubes produced, and ran longitudinally along the tubes just like in the tubes germinated from pollen grains. However, in ungerminated pollen protoplasts, the AFs behaved abnormalty, showing various irregular arrangements. When protoplasts bursted, the actin aggregates often located at the protrusion site from which the protoplasts would burst, and were discharged into the medium. In neither corn pollen nor gladiolus pollen protoplasts AFs were observed within the generative or sperm cells.     

Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.     

Pollen Cryopreservation and Pollen Protoplast Isolation in Brassica carnpestris var.purpurea

The authors have investigated the factors affecting pollen cryopreservation in Brassica campestris var. purpurea, such as pollen development stages cryoprotectant and the process of freezing. A suitable procedure was established as follows :Pollen grains suspended in B5 medium containing 10X DMSO and 1SM sucrose were frozen by a three- –1℃/min – 1 ℃/min step method(0℃———→–10 ℃ ,standing for 15 min———→–40 ℃ , standing for 1 hr→liquid nitrogen)and later thawed in 40℃ water bath. During a period of 60, 90 days′preservation, the relative survival percentage of mature (at the day of anthesis)and nearly mature(2 days before anthesis, trinucleate stage)pollens maintained at ca. 91% that of young pollens(7-8 days before anthesis, late uninucleate stage to early binucleate stage)slightly declined from the original 91.6% to 84. 3%. Culture. experiment showed that the cryopreserved young pollen could be induced to cell division just as well as the fresh pollen. The method of isolating protoplasts from fresh mature pollen developed previously was improved and simplified. As a result, protoplasts were isolated more conveniently from mature pollen and young pollen for the first time. The protoplasts from cryopreserved mature and young pollen could be obtained as well with an isolation rate of 77.4% and 35.9% respectively. However, for isolation of protoplasts from preserved young pollen, an incubation in NLN medium at 35℃ after thawing was necessary.     

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum

Summary A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg^-1 H₂O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca²⁺ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.Abbreviations BCP bromocresol purple - FDA fluoresceindiacetate - MES 2-(N-morpholino) ethanesulfonic acid - PEG polyethyleneglycol     

Isolation and Early in Vitro Development of Young Pollen Protoplasts in Nicotiana tabacum

For isolating young pollen protoplasts in Nicotiana tabacum. The authors had established two efficient enzymatic methods via anther preculture or pollen starvation pretreatment. Procedure of the first method included the following steps: 1. Cold pretreatment of flower buds with pollen at late unicellular to early bicellular stage; 2. Anther floating culture for pollen shedding into the culture medium followed by dehiscence of exine; 3. Enzymatic maceration of exine-dehisced pollen resulting in degradation of intine and release of pollen protoplasts in large quantity. Procedure of the second method involved the following steps: 1. Culture of pollen at middle bicellular in Kyo and Harada' B medium for starvation: 2. Enzymatic maceration of starvated pollen resulting in release of pollen protoplasts and subprotoplasts. Factors affecting the results of both methods as well as early in vitro developmental events of young pollen protoplasts were studied. The protoplasts could be induced either to trigger the first sporophytic division or to continue the gametophytic pathway leading germinatation of pollen tubes !ndicating their potentiality of inducing both sporophytic and gametophytic development of pathway. In rare instance a quite interesting phenomenon was observed that a pollen protoplast first divided into two daughter cells and one of which then germinated a pollen tube. It may insinuate that such pollen protoplasts initially induced a sporophytic pathway could reverse induce a gametophytic pathway.     

Mass Isolation and Preservation of Viable Sperm Cells in Brassica campestris var.purpurea

The two-step osmotic shock and grinding methods reported by Yang and Zhou (1989) were modified for isolation of viable sperm cells in large quantities from pollen grains of Brassica campestris var. purpurea. Factors affecting the yield and survival of isolated sperm cells have been investigated. These included physiological status of donor flowers, sucrose concentration used for pollen hydration, basic media, protectants and osmotica supplemented in the medium etc. As a result, two procedures have been developed. For osmotic shock method, pollen grains at the day of anthesis were hydrated in 25% sucrose solution for 30 min and, after centrifugation and removal of the supernatant, the pellet was shocked by a medium containing 12.5% sucrose, 0.1 g/L KNO3, 0.36 g/L CaCl2, 2H2O, 0.3% potassium dextran sulphate (PDS), 0.6% bovine serum albumin (BSA), and 0.3% polyvinylpyrrolidone (PVP). The viable sperm yield was 34%. After removal of pollen wall debris by filtration and centrifugation, the sperm cell-rich pellets were resuspended in a medium containing 20% sucrose, 5% sorbitol, 0.1 g/L KNOs, 0.36g/L CaCl2·2H2O, 0.6% BSA and 0.3% PDS, and preserved at 4℃ for two days. For grinding method, the pollen grains hydrated in 30% sucrose solution for 30 min. were resuspended in a medium containing 20% sucrose, 5% sorbitol, 0.1g/L QNO3, 0.36g/L CaCl2·2H2O, 0.3% PDS, 0.6% BSA, 0.3% PVP and 20 μg/ml fluorescein diacetate, then ground with a glass homogenizer to release the sperm cells. The viable sperm yield was up to 86%. Following filtration and centrifugation for removal of pollen wall debris, the sperm cells were stored at 4℃ in the same medium but without supplementation of PVP. Tested by fluorochromatic reaction, the sperm cells could survive up to one week with a gradual decline of viability. Cytological observations revealed that pairs of ellipsoidal sperm cells just released were linked together; one of the pair had a long tail-like extension which also show fluorochromasia. Soon after, the sperm cells separated and turned to be spherical. The present results open a prospect to use isolated viable sperm cells for further experimental manipulations.     

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%.     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

Plant Regeneration from Petiole Protoplasts of Brassica napus

Two cultivars of Brassica napus, Altex and Canadian twins, were used as materials. Protoplasts isolated from petioles of plants grown in vitro were cultured in Nitsch medium supplemented with 0.5mg/L BA, 0.5mg/L NAA, lmg/L 2,4-D, 100mg/L serine, 800mg/L glutamine, 4% sucrose and 0.4mol/L mannitol. After 2 days of culture, the first division was observed. The division frequency estimated after 10 days of culture was 30-60%. One week after transferring onto MS medium containing 6mg/L GA3. and 3mg/L BA, protoplast-derived calli regenerated into shoots. The regeneration frequency of the two cultivars was 24% and 31% respectively. It was found that the protoplasts isolated from petioles could float on the surface of the 3% sucrose contained solution which was very favourable both to purification, and culture of the protoplasts.     

Plant Regeneration from Protoplasts of Savoy Cabbage(Brassica oleracea L. var. subauda)

Protoplasts of savoy cabbage (Brassica olleracea L. var. subauda), "SA61" (SV), were isolated from leaves and hypocotyls of seedlings grown in vitro, in enzyme mixture containing 2% cellulase (Onozuka R-10) and 0.8% macerozyme RI0. Good results of protoplast collection were obtained by using 18% and 17% sucrose solution floating leaf protoplasts and hypocotyl protoplasts respectively, and centrifugalizing with the rate of 500 r/min. All the collected protoplasts were cultured in 5 different liquid media from which the best results were observed on DPD1 medium for leaf protoplasts and on MS1 medium for hypocotyl protoplasts, with the highest cell division rate and planting efficiency. About 2 weeks of cultures, many cell clusters and a few embryo-like structures were visualized. The cell clusters developed into visible microcalli in 20-30 days and grew up to 1 mm or so in dimeter about 40 days of culture. For growth, the calli were transferred to 7 different agar media and from which two suitable media, MB2 and MB3, were selected. Cultured for 40-50 days, the calli grew up, and were transferred to 4 solid media for organ differentiation. Ideal results of shoot regeneration were obtained on MS, medium. About 2 weeks after rooted on the MS medium without any auxin, intact plants were regenerated.     

烟草未受精中央细胞及其它胚囊细胞的离体分裂

自70年代中期以来,未传粉子房和胚珠的离体培养已在多种植物中取得成功,得到的单倍体植株来源于胶囊中的卵细胞、助细胞以及反足细胞。而分离的未受精胚囊及其成员细胞的离体培养虽屡经尝试,迄今只有Kranz等诱导了玉米未受精卵细胞分裂形成小愈伤组织,至于中央细胞与其它雌配子体细胞则无离体分裂的报道。本文报道大叶烟草未受精中央细胞首次培养成细胞团及其它胚囊细胞启动离体分裂的实验结果。     

Polyethylene Glycol Induced Fusion of Selected Pairs of Single Protoplasts

A new method for polyethylene glycol (PEG) -induced fusion between single pairs of selected protoplasts was developed. The protoplasts were prepared from tobacco leaves. Under an inverted microscope two defined protoplasts were selected with a hand-made micropipette and transferred into a droplet of fusion solution containing 25 % PEG (M. W. 6000), 0. 1 mol/L mannitol and 0. 01 mol/L CaCl2 · 2H2O (pH 5.6). Slightly moving the pipette caused the protoplasts to contact and adhere to each other, the fusion pairs were then transferred to a solution containing 10% PEG, 0.35 mol/L sucrose and 0. 01 mol/L CaCl2 · 2H2O (pH 5.6) for approximately 10 min, followed by subsequent washing with a solution containing 0.45 mol/L sucrose and 0.04 mol/L CaC12 · 2H20 (pH 7—9). Compared with conventional fusion methods adopted to protoplast population, the present method can avoid either blind fusion of protoplasts belonging to one partner and fusion among multiple protoplasts, or the presence of unfused protoplasts, thus ensure the fusion to be precisely at the level of a selected pair of single protoplasts. Moreover, it is simple and convenient enough to show its potentiality for wide application in somatic hybridization and particularly in the case of small quantity of parental protoplasts such as in vitro intergametic fusion studies.     

The polygalacturonase gene BcMF2 from Brassica campestris is associated with intine development

Brassica campestris Male Fertility 2 (BcMF2) is a putative polygalacturonase(PG) gene previously isolated from the flower bud of Chinesecabbage (Brassica campestris L. ssp. chinensis Makino, syn.B. rapa ssp. chinensis). This gene was found to be expressedspecifically in tapetum and pollen after the tetrad stage ofanther development. Antisense RNA technology was used to studythe function of BcMF2 in Chinese cabbage. Scanning and transmissionelectron microscopy revealed that there were deformities inthe transgenic mature pollen grains such as abnormal locationof germinal furrows. In addition, the homogeneous pectic exintinelayer facing the exterior seemed to be overdeveloped and predominantlyoccupied the intine, thus reversing the normal proportionaldistribution of the internal endintine layer and the externalexintine layer. Since it is a continuation of the intine layer,the pollen tube wall could not grow normally. This resultedin the formation of a balloon-like swelling structure in thepollen tube tip in nearly 80% of the transgenic pollen grains.Premature degradation of tapetum was also found in these transgenicplants, which displayed decreased expression of the BcMF2 gene.BcMF2 might therefore encode a new PG with an important rolein pollen wall development, possibly via regulation of pectin'sdynamic metabolism. Key words: Brassica campestris, Brassica rapa, Chinese cabbage, intine, PG, polygalacturonase, pollen wall Received 28 August 2008; Revised 14 October 2008 Accepted 20 October 2008     

In Vitro Divisions of Unfertilized Central Cells and Other Embryo Sac Cells in Nicotiana tabacum var macrophylla

The embryo sacs and female cells could be isolated from the unfertilized ovules of Nicotiana tabacum L. var. macrophylla which were treated in a solution containing 1.5 % cellulase R- 1O, 1% macerozyme R-10, 10% mannitol, 10 mmol/L CaCI:, pH 5.8 for 3 h followed by given slight pressure with a micropipette. The central cells could be kept viable for 10 h and the egg cells for 3 h in 10% mannital. Sometimes, the in situ fusion products of egg cell and synergid protoplasts could be obtained and kept viable for at least 5 h. The high concentration (20 mg/L) of 2, 4-D was used in enzyme solution to induce the division of the unfertilized central cells and other megagametophytic cells in subsequent culture. Treatment of 2,4-D together with enzymatic maceration of ovules was proved to be better than its direct treatment of isolated embryo sac or its component cells. Isolated embryo sacs were cultured in microchambers (Millicell-CM PICM 012 50 MILLIPORE) feeded with divided mesophyll protoplasts of Nicotiana rustica L. The medium was KMSp medium supple- mented with 1% glucose, 0.1 mol/L mannitol, 0.1 mol/L sorbitol, 0.25 mol/L sucrose, 1 mg/L BA, 6% to 10% coconut water, and 0.15% low gelling agarose. Division of central cells, antipodal cells and the in situ fusion products of egg cell and synergid protoplasts were induced. The unfertilized central cell was for the first time to be induced in vitro to develop into small cell clusters.     

Protoplast Culture and Plantlet Regeneration from Hypocotyls of Brassica campestris var. parachinesis

Protoplasts isolated from 3--4 day-old (ca 4 cm in length) etiolated hypocotyls of Brassica carnpestris var. parachinesis (Bally) Tsen et Lee and purified with 20% sucrose were cultured on K8p medium suplemented with 0. 5 mg/L ZT, 0.5 mg/L 2, 4-D, 1.0 mg/L NAA and 0. 4 mol/L glucose. When initially cultured for 14-18 hours the protoplasts formed new walls and by first division after 36 hours. The divided protoplasts reached 35 % after being cultured for three days. When cultured under optimum conditions for 8-9 days, the proto plasts formed 8-16 cell colonies with a plate effeciency as high as 15%-18%. Rapidly growing and dividing calli of 2 mm in diameter were transferred onto semisold gelrite media with 0.3 mg/L 2, 4-D enabling them to proliferate further towards the size of 4-5 mm in diameter. Shoot differentiation was carried out in MS medium with 3.2 (or 1.6) mg/L BA, 1.6 (or 0.8) mg/L ZT, 0.01 mg/L NAA, 0. 1 mg/L GA3 and 0.2 % sucrose. Shoots were cut down and rooted on medium with 0.2 mg/L IAA and 2 % sucrose where whole plants were evatually developed.     

鹅掌楸属植物花粉萌发前后壁的超微结构

观察描述了在电镜下中国鹅掌楸（Ｌｉｒｉｏｄｅｎｄｒｏｎｃｈｉｎｅｎｓｅ）和北美鹅掌楸（Ｌ．ｔｕｌｉｐｉｆｅｒａ）２种植物花粉壁的超微结构及其水合后的变化。（１）成熟花粉壁由６层组成,即外壁３层──外层,中层１和中层２,内壁３层──内壁１,内壁２和内壁３。（２）花粉水合时,在内壁３与质膜之间由Ｐ一粒子（多糖－粒子）和被膜小泡参与形成新层。（３）花粉萌发时,由内壁３的一部分和新层突出萌发孔共同形成花粉管壁。（４）新层于花粉管形成早期分成２层──外染色深的果胶层和内电子透明的胼胝质层。     

An Ultrastructural Study on Pollen Protoplasts and Pollen Tubes Germinated From Them in Gladiolus gandavensis

Large quantities of protoplasts were isolated enzymatically from the mature pollen grains in Gladiolus gandavensis. Regeneration of cell wall and germination of pollen tubes were performed during culture of purified pollen protoplasts in Ks medium supplemented with 32% sucrose, 0.1 mg/1 2,4-D, 1 mg/1 NAA and 0.2 mg/1 6-BA, with a germination rate up to 47.7%. The materials were fixed gently with gradually increasing concentration of glutaraldehyde, followed by osmium, then preembedded in a thin layer of agar and surveyed under an inverted microscope so as to select desired specimens for subsequent procedure. Small agar blocks containing specimens were dehydrated through ethanal-propylene oxide series, embedded in Araldite and ultratomed. Electron microscopic observations show that the pollen protoplasts are surrounded by a smooth plasma membrane and with ultrastructurally intact cytoplasm, a vegetative nucleus and a generative cell. After 8h of culture, wall regeneration commences resulting in a multilayered, fibrillar wall structure which is different from the intine. No exine is formed. Numerous vesicles participate actively in the wall formation. The wall is uneven in thickness around its periphery; a thickened area somewhat resembling to germ furrow is formed, from which pollen tube emerges. The tubes contain abundant plastids, mitochondria and dictyosomes. Vesicles are released out of the plasma membrane and involved in tube wall formation. After 18h of culture, the vegetative nucleus and generative cell have migrated into the tube. Technical points of preparing pollen protoplast specimens for ultastructural studies and the fearnres of wall regeneration in pollen protoplast culture are discussed.