Evaluation of haploidization efficiency in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) through anther culture

The production of large numbers of haploids is the crucial point of the dihaploidisation process. Although in vitro haploid plants were successfully produced by irradiated pollen technique in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.), the frequency is still insufficient for using in a large-scale breeding programme. Thus, the present study was conducted to determine the efficacy of the anther culture technique on the production of in vitro haploids in the aforementioned species for which there have been no successful reports concerning by androgenesis. The anthers at uninucleate microspore stage were collected at different florescence times and then cultured on a solid MS medium supplemented by different combinations of 2,4-D (2,4-dichlorophenoxyacetic acid), BAP (6-benzylaminopurine), KN (kinetin) with the constant addition of NAA (naphthalene acetic acid) to induce callogenesis, embryogenesis and plantlet initiation. The combination of PGR, genotype and anther collection time played an important role in the androgenic response. The highest response was obtained from 57S?21 and G9 lines with the combination of 2.0 or 4.0 mg/l BAP?+?0.05 mg/l NAA (E6 medium) at the first anther collection time. Plantlets were regenerated and rooted on MS medium supplemented by 0.01 mg/l IAA. In total, 74 plants were recovered and propagated with micro-cuttings. The ploidy analyses revealed that 35 plants (47.3?%) were haploid (n?=?20), and the others (52.7?%) were diploid (2n?=?40).     

采后南瓜果实中几种营养成分的变化

"蜜本"南瓜采后0～150 d期间,其果实中多糖和抗坏血酸含量下降,β-胡萝卜素含量增加;总糖、还原糖和蛋白质含量增加,后期下降;氨基酸含量下降,后期增加.     

Brassinolide-Induced Elongation of Inner Tissues of Segments of Squash (Cucurbita maxima Duch.) Hypocotyls

Brassinolide (BR) stimulated elongation of etiolated squashhypocotyl segments with outer tissues removed, as well as thatof unpeeled segments, while IAA has no effect on peeled segments.BR changed the mechanical properties of cell walls of the innertissue. The inner tissue is probably the target tissue in BR-inducedelongation. ¹Dr. Susumu Kuraishi died in 1993.     

蔓生型南瓜资源部分植物学数量性状的评价探讨

对123份蔓生型南瓜（Cucurbita moschataDuch.）自交系的茎节间长度、主蔓粗、叶片长度、叶片宽度、叶柄长度、叶柄粗、第一朵雌花着生节位、主蔓20节内着生雌花数、花径9个数量性状进行了统计分析,提出节间长度、叶片长度、叶柄长度、第一雌花着生节位和主蔓20节内着生雌花数5个性状可以作为蔓生南瓜植物学性状评价的代表性状,并采用9级评价分级体系将这些代表性状进行评价分级,为建立规范化、标准化的南瓜种质资源评价系统奠定了基础。     

Unchanged Molecular-Weight Distribution of Xyloglucans in Outer Tissue Cell Walls along Intact Growing Hypocotyls of Squash (Cucurbita maxima Duch.) Seedlings

The changes in the mechanical properties and compositions ofcell walls in outer and inner tissues were investigated alongthe hypocotyls of squash (Cucurbita maxima Duch.) seedlings.The endogenous growth capacity decreased and the minimum stress-relaxationtime (T_O) of cell walls in outer tissues increased from theapical to the basal region of hypocotyls. A high correlationwas observed between values of To in outer tissues and endogenousgrowth (r=–0.99). The values of T_O in inner tissues didnot change from the apical to the basal region of hypocotyls. In outer tissues, the levels of neutral sugars in pectin decreasedconsiderably from the apical to the basal region of hypocotyls.However, relative amounts of hemicellulose showed little differencealong the hypocotyls. Levels and molecular weights of hemicellulosicxyloglucans in outer tissues were about 2-3 times greater thanthose in inner tissues. The amount of xyloglucans in outer tissuesincreased in the middle region of hypocotyls, and xyloglucansin upper and basal regions had similar molecular weights. Bycontrast, in inner tissues, amounts of cell-wall material decreasedtoward the basal region. Amounts and molecular weights of hemicellulosicxyloglucans also decreased along the hypocotyls. These results clearly show that cell-wall metabolism duringaging of intact growing stem tissues differs markedly betweenouter and inner tissues, and the absence of a simple relationship between the molecular weights of xyloglucans and the mechanicalproperties of the cell walls in outer tissues indicates thatthe changes in the mechanical properties of the cell walls inintact growing tissues cannot be explained only by the molecularweights of xyloglucans. Thus, the regulation of the mechanicalproperties of cell walls in intact growing stems may be somewhatdifferent from that in auxin-treated stem sections, in whichauxin promotes the depolymerization of xyloglucan molecules. (Received November 28, 1991; Accepted November 16, 1992)     

A Comparison of Stem Peroxidases in Bush and Vine Forms of Squash (Cucurbita maxima Duch.and C. pepo L.)

Total peroxidase activities and peroxidase isozymes were comparedamong 10 bush- and vine-squash cultivars of Cucurbita maximaDuch. and C. pepo (L.) Several qualitative variations of isozymeswere detected between C. maxima and C. pepo, and some differenceswere evident among varieties of C. pepo. There were no qualitativedifferences between genetically similar bush and vine strains.Quantitative differences were difficult to resolve, althoughit did appear that in varieties of C. maxima exhibiting lowlevels of peroxidase activity, isozymes C₃, C₄, and C₆ wereless intense. Depending upon the cultivars compared, total peroxidase activityin bush forms was either higher or lower than, or the same astheir vine counterparts. Since the major bush genes in the varietiesstudied are believed to be allelic, the results indicate thatthe over-all genotype of squash varieties can influence therelative expression of peroxidase activity between bush andvine forms.     

Water Potential and Mechanical Properties of the Cell Wall of Hypocotyls of Dark-Grown Squash (Cucurbita maxima Duch.) under Water-Stress Conditions

Hypocotyl growth of seedlings of dark-grown squash (Cucurbitamaxima Duch.) was greatly reduced by the addition of 60mM polyethyleneglycol (PEG) to hydroponic solution (water stress). Apoplastic solution (A) and cell sap (C) were separately collectedfrom the hypocotyl segments by a centrifugation method. Theosmotic potentials of A (_A) and C (_c), and (=_c–_A) ofstressed hypocotyls were always lower than those of unstressedhypocotyls. Suction force was measured by immersing the segments into solutionsof different concentrations of mannitol. Suction force was significantlycorrelated with _C (r= –0.99). The mechanical properties of the cell wall of hypocotyl segmentswere measured by stressrelaxation technique. Minimum stressrelaxation time (T_o), relaxation rate (R) and residual stressof unstressed hypocotyls were low during the growth period andincreased when the growth ceased. T_o and R of stressed hypocotylsdecreased one day after the stress treatment, but the residualstress was not decreased by the water stress throughout theexperiment. These results suggest that the suppressed growth of dark-grownsquash hypocotyls under water stress was due neither to thereduction of the osmotic potential difference between innerand outer space of the cell, nor to the decrease in suctionforce, but was partly due to the unchanged mechanical propertiesof the cell wall, as represented by one stress-relaxation parameter,residual stress. (Received February 5, 1988; Accepted September 8, 1988)     

Biochemical Characterization and Distribution of Glutathione S-Transferases in Leaping Mullet (Liza saliens)

In this study, feral leaping mullet (Liza saliens) liver cytosolic glutathione S-transferases (GSTs) were investigated and characterized using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as substrates. The average GST activities towards CDNB and EA were found to be 1365 +/- 41 and 140 +/- 20 nmol/min per mg protein, respectively. The effects of cytosolic protein amount and temperature ranging from 4 to 70 degrees C on enzyme activities were examined. While both activities towards CDNB and EA showed similar dependence on protein amount, temperature optima were found as 37 and 42 degrees C, respectively. In addition, the effects of pH on GST-CDNB and -EA activities were studied and different pH activity profiles were observed. For both substrates, GST activities were found to obey Michaelis-Menten kinetics with apparent V(max) and K(m) values of 1661 nmol/min per mg protein and 0.24 mM and 157 nmol/min per mg protein and 0.056 mM for CDNB and EA, respectively. Distribution of GST in Liza saliens tissues was investigated and compared with other fish species. Very high GST activities were measured in tissues from Liza saliens such as liver, kidney, testis, proximal intestine, and gills. Moreover, our results suggested that GST activities from Liza saliens would be a valuable biomarker for aquatic pollution.     

Changes in Wall Polysaccharides of Squash (Cucurbita maxima Duch.) Hypocotyls under Water Stress Condition: II. Composition of Pectic and Hemicellulosic Polysaccharides

Hypocotyl growth of dark-grown squash (Cucurbita maxima Duch.)seedlings was greatly reduced by the addition of polyethyleneglycol (60 mM) to the hydroponic solution through inhibitionof cell elongation. Measurement of the mechanical propertiesof the cell walls revealed that the cell wall of stressed hypocotylswas loosened as much as that of the unstressed hypocotyls, suggestingthat the stressed hypocotyl could not elongate even though thecell wall loosened. Galactose and arabinose in the pectic fraction,which are probably attached to high mol wt rhamnogalacturonans,increased under stressed as well as under unstressed condition.Other polysaccharides including pectic low mol wt galacturonans,hemicellulosic xyloglucans, galactoglucomannans, arabinans,and glucuronoarabinoxylans increased more under unstressed condition.The mol wt of xyloglucans in the hemicellulosic fraction increasedunder unstressed but not under stressed condition. These results suggest that changes in wall structure, such asincreases in high mol wt rhamnogaracturonans rich in arabinoseand galactose residues, and the suppression of polymerizationof xyloglucans are involved in the process of cell wall loosening. (Received December 15, 1986; Accepted June 8, 1987)     

Purification and Properties of 1-AminocycIopropane-l-carboxylate Synthase of Mesocarp of Cucurbita maxima Duch. Fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, which formsAGC from S-adenosylmethionine (SAM), was purified to homogeneityfrom sliced and aged mesocarp tissue of Cucurbita maxima Duch.cv Ebisu fruits, and its enzymatic properties were determined.The specific activity of the purified enzyme was 220 mU/mg proteinat 30°C at 50 µM SAM. Native ACC synthase has a relativemolecular mass of 160 ± 10 kDa and consisted of two subunitsof about 84±3 kDa. S-adenosylhomocysteine (SAH), S-methylmethionine(SMM) and L-methionine did not serve as substrate. The enzymereaction was competitively inhibited by aminoethoxyvinylglycine(AVG) (Ki, 2.5 µM), aminooxyacetic acid (Ki, 40 µM)and SAH (Ki, 30 µM). The reaction was also strongly inhibitedby semicarbazide, and less effectively by homocysteine. Theenzyme was rapidly inactivated by its substrate, SAM in thepresence of pyridoxalphosphate (PLP), but in the absence ofPLP, SAM-induced inactivation was much slower. Inactivationdid not occur by SAH and SMM, SAM analogs without substrateactivity. Pyridoxal phosphate was an essential cofactor to beadded to a reaction mixture for maximum activity, but an enzymepreparation from which pyridoxal phosphate was removed by SephadexG-25 gel filtration exhibited one-eighth activity which wasinhibited by semicarbazide, this indicating that a small amountof pyridoxal phosphate is firmly bound to the enzyme. (Received May 6, 1986; Accepted May 20, 1986)     

Auxin-stimulated ATPase in membrane fractions from pumpkin hypocotyls (Cucurbita maxima L.)

Membrane fractions from Cucurbita maxima hypocotyls were isolated in a medium which inhibits the action of endogenous phospholipases. After removal of soluble phosphatases by Sepharose 2B-CL column chromatography, an auxin-stimulated ATPase activity was found in membrane fractions from linear sucrose gradients. In the presence of 10^-4 M phenylacetic acid (PAA), the stimulation by indol-3-acetic acid (IAA) exhibited a bimodal concentration dependence with maximal stimulation of about 50% at 10^-6 M IAA. Without PAA, only a high concentration of 10^-4 M IAA was stimulatory, whereas 10^-6 M IAA had no apparent effect and 10^-8 M IAA exhibited weak inhibition. PAA alone had only weak or no effects. The effects of IAA must be considered as hormone-specific. The ATPase activity in the presence of 10^-4 M PAA was activated only by 2,4-dichlorophenoxyacetic acid (2,4-D), an active auxin analogue, but not by the inactive stereoisomers, 2,3-D and 3,5-D. Comparison with marker enzyme profiles suggested that part of the auxin-stimulated ATPase was localized on plasma membranes as well as other compartments. Thus, the auxin-stimulated ATPase may become a useful tool in the investigation of the mechanism of action of auxin.Abbrevations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,3-D 2,3-dichlorophenoxyacetic acid - 3,5-D 3,5-dichlorophenoxyacetic acid - IAA indol-3-acetic acid - PAA phenylacetic acid - MES (2-(N-morpholino))-ethanesulfonic acid - EDTA ethylenediamine tetraacetic acid     

不同方法整枝后的南瓜果实成熟过程中果胶、戊糖和铬(Cr3+)含量的变化

单干、双干和三干3种整枝方式下的南瓜‘龙圆栗香’果实成熟过程中果胶含量在总体上均呈现先高后低的变化趋势。其中,单干和双干整枝的果实中,果胶含量先是急剧升高,30d后急剧下降,而三干整枝的变化趋势比较平缓。果实成熟过程中的戊糖和铬(Cr3 )含量变化趋势大体一致,都呈上升趋势。其中,双干和单干整枝的上升趋势明显。双干整枝的果实中果胶、戊糖和铬(Cr3 )含量都一直是最高。     

Pumpkin (Cucurbita sp.) seed globulin II. Alterations during germination

Alterations in pumpkin seed globulin during germination wereexamined mainly by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. The total protein content of etiolated cotyledonsdecreased to 32% of the initial content 14 days after germination,and the globulin was rapidly degraded to produce new componentswith molecular weights of about 40,000 (major component) and30,000 daltons, which were soluble in a solution with relativelylow concentrations of salt at neutral pHs in contrast to insolublecrystalline globulin. The proportion of these soluble proteinsto the total amount of protein extracted in 2 M NaCl solutionincreased rapidly during the period of 2 to 4 days after planting.During this period, the globulin was rapidly degraded with progressiveincrease of the component of 40,000 daltons. This species wasseparated into two polypeptide chains with molecular weightof about 20,000 daltons by treatment with sulfhydryl reducingreagent. The soluble fraction preserved oxaloacetate decarboxylaseactivity. These changes in solubility and subunit structure,and the preservation of the decarboxylase activity of heat stableproteins obtained from etiolated cotyledons are discussed. (Received April 9, 1976; )     

四倍体南瓜的诱变和特性研究

以中国南瓜种‘上海小闸黄狼’南瓜为试材,用秋水仙素为诱变剂,对‘黄狼’种子进行处理,得到了同源四倍体南瓜。在4种浓度处理4h后,发现获得同源四倍体的最佳浓度为0．2％和0．3％。变异材料染色体数加倍（2n=4x=80）。研究发现,与二倍体相比,四倍体单位面积叶面气孔数变少,叶色变深,叶裂变浅,叶尖变钝,叶变大,茎变粗,大量出现畸形雄花,果实变小,果形变直,果柄变短,单瓜种子平均数由340粒减为68粒,抗早衰性更强,脯氨酸含量和过氧化物歧化酶活性更高,丙二醛含量更低。四倍体南瓜伤流量明显比二倍体高,根系活力强于二倍体。     

Sugar Composition and Molecular Weight Distribution of Cell Wall Polysaccharides in Outer and Inner Tissues from Segments of Dark Grown Squash (Cucurbita maxima Duch.) Hypocotyls

The elongation growth of stem segments is determined by the outer cell layers (epidermis and collenchyma). We measured the sugar composition and molecular weight distribution of pectin and hemicellulose fractions obtained from inner and outer tissues of squash (Cucurbita maxima Duch.) hypocotyls. In addition, we studied the changes in these parameters after a 9 hour period of incubation of the segments. The results show that outer tissues have higher molecular weight pectin and hemicellulose compared to inner tissues (2-3 times higher). Incubation results in a 13 to 25% decrease in the amount of pectin and hemicellulose in inner tissues and an increase of 11 to 32% in the outer tissues. This increase in the outer tissues is accompanied by a decrease in the molecular weight of some of the components. These results clearly show that cell wall metabolism during elongation growth differs markedly in inner and outer tissues, and that future studies on the effect of auxin need to take these differences into account.     

Purification and Characterization of Class Alpha and Mu Glutathione S-Transferases From Porcine Liver

Six cytosolic GSTs from porcine liver were purified by a combination of glutathione affinity chromatography and ion-exchange HPLC. The isoenzymes were characterized by SDS-PAGE, gel filtration, isoelectric focusing, immunoblotting analysis and determination of substrate specificities and inhibition characteristics. The purified GSTs belong to the alpha and mu classes, respectively. No class pi isoenzyme was isolated or detected. The class alpha GST pA1-1* exists as a homodimer (M_r = 25.3 kDa), whereas GST pA2-3* consists of two subunits with different M_r values (27.0 and 25.3 kDa). The estimated pI values were 9.5 and 8.8, respectively. Furthermore, four class mu porcine GSTs, pM1-1*, pM1-2*, pM3-?* and pM4-?*, were isolated. The isoenzyme pM1-1* possesses a relative molecular mass of 27.2 kDa and a pI value of 6.2. Additional pM1 isoenzymes hybridize with the subunit pM2* (M_r = 25.2) to furnish a heterodimer, which shows a pI value of 5.8. The other class mu isoenzymes are heterodimers with pI values of 5.45 and 5.05. Substrate specificities and inhibition characteristics correlate very well with those of the corresponding human isoenzymes. The results are discussed with regard to the usefulness of porcine GSTs as an in vitro testing model.     

印度南瓜梭形果突变体的发现及遗传分析

从印度南瓜自交系HL中发现了一个梭形果突变体HL-fu。该突变体的花、果、茎等多器官的生物学特性同时发生了明显变异:雌花及雄花花器中异性花蕊的残存度较野生型高;果实为梭形,果形指数达野生型5倍;节间呈现长短交替分布的排列状态,叶序类似于对生。以突变体HL-fu与野生型HL杂交分别构建了正、反交F1,正交F2,BC1(P1)F1、BC1(P2)F1群体进行遗传分析,推测该变异类型为隐性多效单基因突变,突变性状受1对隐性核基因fu控制。探讨了梭形果突变体在南瓜育种及相关基础研究方面的潜在价值。