黑莓外植体褐化影响因素分析及适宜培养条件筛选

以黑莓(Rubus spp.)品种'Kiowa'为实验对象,研究了基本培养基类型、外植体类型、枝条暗处理时间、防褐化剂以及光照度对组织培养中外植体褐化及腋芽诱导的影响.结果表明,在供试的6种基本培养基(MS、1/2 MS、B5、1/2 B5、N6和1/2 N6)中,在MS培养基上外植体的褐化率较低(37.78%)、腋芽诱导率最高(67.89%)且生长状况良好;褐化程度与外植体的木质化程度及取材部位均有一定的关系,以1年生半木质化枝条的上部茎段为外植体,褐化率最低(19.67%)、腋芽诱导率较高(91.89%)且生长状况良好;接种前进行一定时间的暗处理对外植体褐化有一定的抑制作用,对枝条暗处理3 d,防褐化效果最佳;在培养基中添加不同类型和浓度的防褐化剂对外植体褐化和腋芽诱导也有一定的影响,以添加3 000 mg·L-1活性炭或200 mg·L-1 VC的防褐化效果较好;培养过程中的光照度与外植体褐化也有一定的关系,在1 500 lx光照度下外植体的褐化率较低(15.66%)且腋芽诱导率和生长状况均较好.根据实验结果,筛选出防止黑莓外植体褐化的适宜培养条件为:以1年生半木质化枝条的上部茎段为外植体,15 ℃暗处理3 d后,消毒并接种在添加了3 000 mg·L-1活性炭或200 mg·L-1 VC的MS培养基(含20 g·L-1蔗糖、5.6 g·L-1琼脂及0.2 mg·L-1 6-BA,pH 5.8)上,置于光照度1 500 lx的条件下培养.     

黑莓品种‘Arapaho’离体叶片不定芽诱导影响因素分析及再生体系建立

以黑莓(Rubus spp.)品种‘Arapaho’无菌苗叶片为外植体,通过正交和单因素实验分别研究了基本培养基类型、6-BA和1BA质量浓度以及暗培养时间、外植体的叶位和接种方式对不定芽诱导的影响,并研究了IBA质量浓度对不定芽生根的影响;在此基础上,初步建立了黑莓品种‘Arapaho’离体叶片的再生体系.正交实验结果表明:基本培养基类型对叶片不定芽诱导率及平均不定芽数的影响最大,而IBA质量浓度对叶片不定芽诱导率及6-BA质量浓度对平均不定芽数的影响较小;适宜‘Arapaho’叶片不定芽诱导的最佳培养基为含有2.0mg·L-16-BA和1.0 mg·L-1IBA的MS培养基.单因素实验结果表明:暗培养时间、外植体的叶位及接种方式对不定芽诱导率有显著影响;最适宜的暗培养时间为21 d;植株中、上部叶片的再生能力较强,其中第3和第4位叶的不定芽诱导效果最佳;叶面朝上接种更有利于不定芽的诱导.在含0.2 mg·L-1 IBA的MS培养基中,不定芽生根率达100.0％,且根数多、长势良好.黑莓品种‘Arapaho’离体叶片的再生体系为:以无菌苗的第3和第4位叶为外植体,经过适当修剪后叶面朝上接种于含有2.0 mg·L-16-BA和1.0 mg·L-1IBA的MS培养基上,暗培养21 d后置于光照条件下培养30 d;将不定芽转接到含有0.5 mg·L-16-BA和0.3mg·L-1 NAA的MS培养基上进行继代培养;当不定芽高约2 cm时转接到含有0.2 mg·L-1IBA的MS培养基上进行生根培养,最终获得完整植株.     

苦瓜离体培养过程中内源激素含量的变化

针对苦瓜（Momordica charantia）离体培养中外植体易于产生愈伤组织而难以再生不定芽的问题,本文采用酶联免疫吸附法,研究了苦瓜组织培养过程中不同发育阶段各外植体内源激素含量的变化,以探讨不定芽分化与激素水平变化之间的关系,以及苦瓜难以再生不定芽的内在制约因素。结果表明：（1）苦瓜外植体中LAA含量较高,而iPAs含量过低,是苦瓜易于产生愈伤组织而不定芽再生困难的主要原因;（2）不定芽的分化与IAA／iPAs的变化有密切的关系;（3）在离体培养过程中,保持外源细胞分裂素类物质（如ZT）的适当浓度并及时继代,有利于苦瓜不定芽的分化。     

苦瓜离体培养过程中内源激素含量的变化

针对苦瓜(Momordica charantia)离体培养中外植体易于产生愈伤组织而难以再生不定芽的问题,本文采用酶联免疫吸附法, 研究了苦瓜组织培养过程中不同发育阶段各外植体内源激素含量的变化, 以探讨不定芽分化与激素水平变化之间的关系, 以及苦瓜难以再生不定芽的内在制约因素。结果表明: (1)苦瓜外植体中IAA含量较高, 而iPAs含量过低, 是苦瓜易于产生愈伤组织而不定芽再生困难的主要原因;(2)不定芽的分化与IAA/iPAs的变化有密切的关系; (3)在离体培养过程中, 保持外源细胞分裂素类物质(如ZT)的适当浓度并及时继代, 有利于苦瓜不定芽的分化。     

黑莓品种‘Boysen’果实水提物和色素对DPPH·的反应动力学特性及清除能力研究

以VC和BHT为对照,运用反应动力学研究方法对黑莓(Rubus spp.)品种‘Boysen’果实的水提物和色素与1,1-二苯基-2-苦基肼自由基(DPPH.)的反应动力学特性和清除能力进行了研究。结果表明:VC、BHT、黑莓果实水提物和色素与DPPH.反应完全所需的时间差异较大,其中,0.05～0.26 g·L-1VC仅为1 min,0.51～4.04 g·L-1BHT超过120 min,10.00～100.00 g·L-1水提物为28～100 min,0.03～0.21 g·L-1色素为10～100 min;VC与DPPH.的反应在瞬间完成,BHT与DPPH.的反应可分为2个阶段,而水提物或色素与DPPH.的反应均可分为3个阶段,各阶段的反应速率常数(k)值均随反应时间而减小;随VC、BHT、水提物和色素质量浓度的提高,与DPPH.的反应速率均逐渐增加,整体反应时间缩短;总体上,水提物和色素与DPPH.的反应速率比VC低,但明显高于BHT,k值由大至小依次为VC、色素、水提物、BHT。黑莓果实水提物清除DPPH.的半数有效浓度(EC50)为20.0 g·L-1,高于BHT和VC;总抗氧化能力值(TCA)和清除自由基能力(AE)分别为2.89μmol和4.99×10-4,均低于BHT和VC,说明黑莓果实水提物的抗氧化特性比BHT和VC弱。黑莓果实色素的EC50为7.16×10-2 g·L-1,低于BHT和VC;TCA为809μmol,高于BHT和VC;AE为1.40×10-1,低于VC但明显高于BHT,说明黑莓果实色素的抗氧化特性比BHT强。研究结果显示:黑莓果实色素是一种优良的天然抗氧化剂,值得进一步开发应用。     

金花茶子叶在离体培养中胚状体的发生和小植株的形成

研究了金花茶(Camellia chrysantha(Hu)Tuyama)子叶在离体培养中体细胞胚状体发生的条件。在MS基本培养基中附加苄基嘌呤(BA)或苄基嘌呤与萘乙酸(NAA)组合,诱导了胚状体发生。组织学观察表明,胚状体起源于子叶的表皮细胞。在增添细胞分裂素和生长素的MS或改良B_5液体培养基里振荡培养,明显地促进了胚状体根的生长和茎的发育。胚状体在继代培养中能保持旺盛的再生能力。已得到两个繁殖率较高的胚状体无性系。在合适的条件下,胚状体能长成正常的小植株。     

催吐萝芙木离体培养和植株再生体系的建立

为建立催吐萝芙木(Rauvolfia vomitoria Afzel.)的快繁再生体系,以茎段为外植体,比较了植物生长调节剂对其愈伤组织诱导、分化及生根的影响。结果表明,诱导愈伤组织的适宜培养基为MS+2,4-D 1.0 mg L~(–1)+TDZ 0.5 mg L~(–1)或MS+2,4-D2.0 mg L~(–1)+TDZ 0.5 mg L~(–1),出愈率达100%且生长状况良好;诱导丛生芽的最佳培养基为MS+6-BA 3.0 mg L~(–1)+NAA 0.1 mg L~(–1),出芽率为46.6%,平均出芽数为3.04。这为催吐萝芙木的快速繁殖和遗传转化研究奠定了基础。     

‘杨氏金红50号’猕猴桃的离体快繁研究

为建立‘杨氏金红50号’猕猴桃的离体快繁体系,该研究以其带腋芽茎段为外植体,采用组织培养的方法进行离体培养,并建立了两种离体再生途径:途径I为直接诱导茎段腋芽出芽;途径Ⅱ为茎段基部先产生愈伤组织,再分化不定芽。结果表明:‘杨氏金红50号’猕猴桃带腋芽茎段的最佳灭菌方式为75%酒精30 s+15%Ca(Cl O)_25 min+0.1%升汞8 min;在途径I中,诱导茎段腋芽出芽的最优培养基为MS+4.0 mg·L~(-1)6-BA+0.1 mg·L~(-1)NAA;在途径Ⅱ中,诱导茎段基部产生愈伤组织并产生不定芽的最优培养基为MS+3.0 mg·L~(-1)6-BA+0.3 mg·L~(-1)NAA;培养丛生芽的最佳植物生长调节剂组合为MS+4.0 mg·L~(-1)6-BA+0.4 mg·L~(-1)NAA;不定芽生根培养的最佳植物生长调节剂组合为1/2 MS+0.9 mg·L~(-1)IBA,20 d左右分化出不定根,40 d左右获得完整植株;生根后的组培苗在田园土∶细沙=1∶1的基质中能达到96%的移栽成活率。     

蛇床幼茎离体培养中体细胞胚胎形成的观察

蛇床幼茎外植体经诱导产生了愈伤组织。在ＭＳ＋２,４－Ｄ,０．２ｍｇ／Ｌ＋ＺＴ０．４ｍｇ／Ｌ培养基中,愈伤组织转变成胚性愈伤组织。转入ＭＳ＋ＮＡＡ０．２ｍｇ／Ｌ＋ＺＴ０．８ｍｇ／Ｌ培养基以后,胚性愈伤组织分化出体细胞胚胎。体细胞胚胎在ＭＳ＋ＮＡＡ０．５ｍｇ／Ｌ培养基中可直接发育成为完整植析。显微观察表明,体细胞胚胎产生于愈伤组织的表层细胞或内部细胞。在鱼雷胚期已有螺纹导管的分化。子叶期的维管组织从两     

矮生羽扇豆品种‘画廊’的组织培养和快速繁殖

1植物名称羽扇豆矮生品种‘画廊’（Lupinhis polyphyllus Lindl cv．‘gallery’）。2材料类别种子、根茎。3培养条件种子萌发培养基：（1）MS;生根培养基：（1）MS,（2）1／2MS,（3）MS＋NAA0．1mg·L-1（单位下同）;增殖培养基：（4）MS＋6-BA0．1＋NAA0．01,（5）MS＋6-BA0．2＋NAA0．02,（6）MS＋6-BA0．5＋NAA0．05。培养基中附加0．6％琼脂和3％蔗糖,pH5．8。     

黑莓品种'Boysen'鲜果贮藏特性的研究

对黑莓(Rubus spp. )品种'Boysen'不同成熟度(七成熟、八成熟和九成熟)的鲜果在常温(25 ℃)和低温(4 ℃)条件下的贮藏特性进行了研究,对贮藏时间以及贮藏前后果实的感官指标(色泽、香味和霉烂指数)、形态指标(质量和平均果径)及品质指标(可溶性固形物含量、可滴定酸含量、总花色苷含量以及固酸比)进行了比较.结果表明,不同成熟度和贮藏温度对黑莓鲜果的贮藏时间有明显影响,常温条件下九成熟、八成熟和七成熟果实的贮藏时间分别为3、4和5 d,低温条件下则分别为7、9和14 d;果实霉烂指数则随成熟度的提高及贮藏时间的延长不断提高,成熟度或贮藏温度越低(大于0 ℃),霉烂指数越小,果实的贮藏时间就越长;七成熟和八成熟的果实贮藏后色泽加深、香味变浓,而九成熟果实贮藏前后色泽和香味变化不大.采摘时不同成熟度的果实形态指标和品质指标具有一定的差异,其中,果实成熟度越大,果实质量、平均果径、可溶性固形物含量、固酸比及总花色苷含量越高,可滴定酸含量越低.低温及常温下贮藏后果实质量及平均果径、可溶性固形物含量和可滴定酸含量均不同程度减少,而固酸比和总花色苷含量均不同程度提高,但在低温贮藏条件下各项指标的变化幅度明显小于常温.总体上,在低温条件下,八成熟果实的霉烂指数低,贮藏时间较长,贮藏后的果实质量和平均果径变化幅度小,可溶性固形物含量、可滴定酸含量和总花色苷含量以及固酸比逐渐接近九成熟果实,果实品质提高.因而,在实际生产中宜采收八成熟果实并在4 ℃条件下贮藏和运输,能较好地保持黑莓鲜果的品质.     

DNA fingerprint variation in some blackberry species (Rubus subg.Rubus,Rosaceae)

We have analysed samples from Sweden, Denmark, and Germany of six facultatively apomictic blackberry species to investigate the accordance between a taxonomy based on morphological characters on the one hand, and distribution of genetic variation estimated by DNA fingerprinting on the other hand. DNA fingerprint variation was found to be quite restricted in all species investigated. The first taxonomic group included three species related toRubus nessensis, two being characterized by one very widespread DNA fingerprint in all three countries and a few rare ones, whereas the third species differed between Sweden and Germany. The second taxonomic group included species related toR. gracilis. Two of these species exhibited very similar DNA fingerprints, whereas the third species deviated clearly. The utilization of DNA fingerprinting as a tool in taxonomy is discussed; most likely this method could become a useful complement to morphology, especially in plant groups with reduced levels of genetic recombination.     

Fruit abscission and ethylene production of four blackberry cultivars (Rubus spp.)

In all cultivars the force required to harvest fruit declined during ripening. The fruit retention strength (FRS) of ripe fruit varied between cultivars with ‘Ashton Cross’ and ‘Chehalem’ being easier to remove than ‘Bedford Giant’ or ‘Oregon Thornless’. The cultivars ‘Ashton Cross’ and ‘Oregon Thornless’ showed no increase in ethylene production during fruit ripening whereas ‘Bedford Giant’ and ‘Chehalem’ had increased rates of ethylene production (EPR) in the ripe fruit. Exogenous ethylene accelerated abscission, ethylene production and pigment changes in ‘Ashton Cross’ fruit at all stages of development. 1-amino-cyclopropane-1-carboxylic acid (ACC) supplied to fruit at all stages of development was converted to ethylene at levels in excess of those found naturally. The differences between cultivars are discussed with reference to the role of ethylene in both machine harvesting and post-harvest storage of blackberry fruit.     

Pollen stainability variation in the apomictic blackberry, Rubus nessensis (Rosaceae)

Pollen quality was assessed with cotton blue staining in an apomictic blackberry, Rubus nessensis W. Hall, on plants grown outdoors in a random arrangement under uniform conditions. Significant variation between populations may be attributed mainly to genetical factors. Most of the variation found between plants, sometimes even between ramets, within a population must, however, be environmentally caused, likewise the variation between flowers within the same plant.     

Flowering and fruiting phenology in the apomictic blackberry, Rubus nessensis (Rosaceae)

Five stands of an apomictic blackberry, Rubus nessensis , were studied. Biennial shoots flower and fruit in their second year on lateral branches developed from axillary buds. Most premature death of reproductive units occurs by withering of entire laterals, presumably due to damage by frost during the preceeding autumn or winter. Health status of the floricane, assessed as percentage live axillary buds in the spring, was positively correlated with the reproduction parameters. Shoot height as well, is positively correlated with number of flower buds and flowers produced on each lateral but, however, negatively correlated with number of berries, probably because tall shoots tend to suffer more damage by frost which seriously afflicts the vascular tissues of the laterals.
The position of the axillary buds on the shoot affects survival and degree of fertility, those situated medially greatly surpassing apical and basal ones.     

A demographic study of the apomictic blackberry, Rubus nessensis (Rosaceae)

Ten stands of Rubus nessensis , an apomictic blackberry, were studied. Perennial stools produce biennial shoots that flower in their second year. The stools tend to produce shoots of similar height for several consecutive years. Flowering, but not relative fruit set, is dependent on shoot height. Shoot height and density appear to be positively related, implying that intraspecific competition is of little importance for plant vigour. Depletion time varies greatly between stands (4–29 years), being comparatively longer in stands with short shoots. New establishment is sparse and takes place mostly by root suckering.     

Sugar beet (Beta vulgaris L.) morphogenesis in vitro: effects of phytohormone type and concentration in the culture medium, type of explants, and plant genotype on shoot regeneration frequency

In vitro regeneration techniques have been optimized for seven strains and cultivars of sugar beet (Beta vulgaris L.) bred in Russia. The frequency of shoot regeneration from somatic cells and tissues of sugar beet varies from 10 to 97% depending on the explant type, culture-medium composition, and genotype. The in vitro regeneration potential has been estimated in plants with different genotypes. The effect of medium composition (phytohormones and carbohydrates) on the frequency of the formation of a morphogenic callus competent for plant regeneration has been determined. The effect of the types and concentrations of various cytokines (zeatin, kinetin, and 6-benzylaminopurine) on direct shoot regeneration from cotyledon nodes has been estimated. The culture-medium composition has been optimized for direct shoot regeneration from petioles. The effects of different concentrations of abscisic acid on the frequency of shoot regeneration from a morphogenic callus has been studied. Micropropagation has been used to obtain petiole explants and reproduce the shoots obtained by direct regeneration from cotyledon nodes, petioles, and calluses. Improved shoot-regeneration methods can be used for both agrobacterial and bioballistic genetic transformation of the sugar beet genotypes studied.     

Sugar beet (Beta vulgaris L.) morphogenesis in vitro: Effects of phytohormone type and concentration in the culture medium, type of explants, and plant genotype on shoot regeneration frequency

In vitro regeneration techniques have been optimized for seven strains and cultivars of sugar beet (Beta vulgaris L.) bred in Russia. The frequency of shoot regeneration from somatic cells and tissues of sugar beet varies from 10 to 97% depending on the explant type, culture-medium composition, and genotype. The in vitro regeneration potential has been estimated in plants with different genotypes. The effect of medium composition (phytohormones and carbohydrates) on the frequency of the formation of a morphogenic callus competent for plant regeneration has been determined. The effect of the types and concentrations of various cytokines (zeatin, kinetin, and 6-benzylaminopurine) on direct shoot regeneration from cotyledon nodes has been estimated. The culture-medium composition has been optimized for direct shoot regeneration from petioles. The effects of different concentrations of abscisic acid on the frequency of shoot regeneration from a morphogenic callus has been studied. Micropropagation has been used to obtain petiole explants and reproduce the shoots obtained by direct regeneration from cotyledonnodes, petioles, and calluses. Improved shoot-regeneration methods can be used for both agrobacterial and bioballistic genetic transformation of the sugar beet genotypes studied.     

黑莓杂交后代单株主要生长及结果性状的变异分析

对黑莓(Rubus spp.)6个品种9个杂交组合(‘Chester’בKiowa’、‘Kiowa’בNavaho’、‘Triple Crown’בKiowa’、‘Kiowa’בHull’、‘Triple Crown’בChester’、‘Arapaho’בHull’、‘Navaho’בKiowa’、‘Hull’בKiowa’和‘Kiowa’בKiowa’)的131株后代实生苗的主要生长性状(包括刺密度、叶绿素相对含量、分枝数量、主枝和侧枝的直径以及自然株高)和结实性状(包括结果株数、单株果穗数量、每穗果实数量和单株果实数量)进行了观测,并对这些性状的变异状况进行了比较分析.结果表明:在获得的131株杂交后代单株中,有92株具刺植株和121株结果植株,具刺和结果植株的数量分别占后代总株数的70.23％和92.37％.131株杂交后代刺密度、分枝数量、主枝直径、侧枝直径和自然株高的平均值分别为1.51 cm-1、15.43、1.31 cm、0.74 cm和61.07 cm,变异系数分别为78.81％、52.69％、25.19％、20.27％和38.91％;121株杂交后代结果单株的果穗数量、每穗果实数量和单株果实数量的平均值分别为27.05、4.92和145.98,变异系数分别为67.63％、50.83％和91.20％;在不同杂交组合间后代单株的这些指标变异均较大,且与亲本相比,杂交后代的枝蔓生长量和结果数量退化明显且分离严重.杂交后代叶片叶绿素相对含量(Spad)的平均值为45.36,变异系数仅为1.46％,变异幅度较小且与亲本的差异不明显,说明黑莓叶片叶绿素含量性状的遗传力较高且分离小.在无刺品种‘Triple Crown’与具刺品种‘Kiowa’杂交组合的40株杂交后代单株中具刺单株比例达到90％,且其他具刺亲本杂交组合的后代也多数具刺,表明黑莓的刺性状为显性遗传且具有数量遗传特征.