外源细胞分裂素对玉米叶原基分化类型的调控

以互生玉米自交系H4d、8701d为材料,取自交授粉12 d的幼胚,分别在含不同浓度6-BA的脱分化培养基上诱导愈伤组织,筛选II型愈伤组织,分别转入0~3 mg L1 6-BA浓度梯度的分化培养基上诱导出芽。芽点出现时,在解剖镜下剥离顶端分生组织,电镜扫描观察叶原基形态,并用ELISA反应测定顶端分生组织细胞分裂素6-BA、ZRs、DHZRs、iPAS和生长素IAA、NAA含量。结果表明,外源细胞分裂素6-BA可调控互生基因型玉米材料幼苗叶原基转换为对生甚至畸形叶原基类型,6-BA浓度越高、处理越早,出现对生叶原基、畸形叶原基的频率越高。ELISA反应显示,生长点激素含量的改变是外源激素的吸收累积造成的,内源细胞分裂素的含量并未明显改变,在互生基因型下通过增加外源细胞分裂素6-BA提高细胞分裂素/生长素值可导致叶原基分化类型向对生、畸形类型转换,解除外源激素作用后,生长点细胞分裂素/生长素值的恢复导致叶原基分化类型返回原始基因型类型。这进一步证实生长点细胞分裂素/生长素是决定玉米叶原基分化类型的关键因素。     

Genetic variation and control of plant regeneration in leek (Allium ampeloprasum L.)

Zygotic embryos of leek (Allium ampeloprasum L.) were isolated from mature seeds of different cultivars, selfings and full-sib families. The embryos were cultured on callus induction and shoot regeneration medium and employed to study several parameters: percentage of embryos forming calluses, percentage of embryos forming compact calluses, callus weight, percentage of regenerating calluses, numbers of shoot primordia and numbers of regenerated shoots. Differences between cultivars and selfings were found for most parameters studied. For all cultivars all parameters, except callus weight, decreased after one generation of selfing. Compact callus types enhanced primordia formation and shoot regeneration. Genetic characteristics of callus development and plant regeneration were studied in a 4 × 4 diallel cross. Analysis of variance revealed significant differences between full-sib families. The diallel analysis showed that additive gene effects were significant for all parameters. The predominance of additive gene effects indicated high narrow-sense heritability. Breeding for an increased number of regenerated shoots was successful.     

Improved plant regeneration and in vitro somatic embryogenesis of St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze]

St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is an important warm season turf and pasture grass. In vitro tissue culture of St Augustinegrass could serve as an important mean for its improvement through genetic transformation as well as induced somaclonal variation. To optimize tissue culture conditions for plant regeneration of St Augustinegrass, tissue culture responses of 11 explant tissues and four callus induction/subculture media have been examined. Embryogenic calli with regeneration potential were observed on cultures of early immature embryo [3 days after pollination (DAP)], immature embryo (7–14 DAP), and shoot base of young seedlings. The addition of benzyladenine (BA) in the callus induction/subculture medium enhances callus regeneration ability and does not harm callus induction for immature embryos. The best response came from 7 to 14 DAP immature embryo on MS medium containing 1 mg/l 2,4‐dichlorophenoxyacetic acid and 0.5 mg/l BA. The callus induction and regeneration rates were 97.7% and 47.6% respectively. However, BA supplement reduced callus formation and failed to enhance regeneration for young leaf bases. Scanning electron microscopy revealed that plant regeneration of St Augustinegrass is via somatic embryogenesis.     

A rapid modification of de Lautour's technique for grass leaf chromosomes

A modification of de Lautour's technique for grass leaf chromosomes is described which makes possible rapid sampling and bulk processing of grass shoot tissues for cytological examination. These tissues have a number of advantages over the root-tip tissues normally used for this purpose, of which the most important is that the supply of suitable cells in division is independent of season.     

Electrophoretic patterns of acid soluble proteins and active isoforms of peroxidase and polyphenoloxidase typifying calli and somatic embryos of two reputed date palm cultivars in Morocco

Summary When subjected to micropropagation by tissue culture, the two reputed cultivars of date palm (Phoenix dactylifera L.); Bou-Sthammi noire, resistant to Bayoud disease and Bou-Feggous, of high fruit quality, give rise to three types of calli, called white and root-forming callus, hyperhydric and degenerating callus and friable and embryogenic callus. All explant sources, calli and germinated embryos were analysed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) for acid soluble protein composition. Phenol-oxidizing enzymes; peroxidase and polyphenoloxidase, were also, evaluated and the isoforms separated by polyacrylamide gel electrophoresis. When compared with the explant and germinated embryos, embryogenic calli of the two date palm cultivars could be identified by a concentrated polypeptide of molecular weight 27 500 and polypeptides of molecular weights 70 000 and 11 500. Hyperhydric and degenerating callus contained the polypeptide exhibiting the molecular weight 32 000. Embryogenic calli showed high levels of soluble, ionically and covalently bound peroxidases. The soluble acidic isoperoxidase of R _f 0.60, revealed in these calli and germinated embryos could be a marker of the two tissues. White and root-forming calli of Bou-Feggous cultivar were typified by soluble acidic isoperoxidases with high mobility (R _f 0.75) and anodic ionically wall-bound polyphenoloxidases similar to those of the explant sources. Polyphemoloxidase activities detected in calli and embryos were very low when compared with those of explants. Used as an early test to screen embryogenic calli of date palm, acid soluble proteins, peroxidase and polyphenoloxidase data could lead to introduce lightening and economy in the tissue culture technique.     

Medium, Explant and Genotype Factors Influencing Shoot Regeneration in Oilseed Brassica spp.

The effects of culture media, explants and genotypes on shoot regeneration in oilseed Brassica species were examined in this study. The maximum shoot regeneration frequency was obtained in Murashige and Skoog medium supplemented with 3 mg l^?1 6‐benzylaminopurine and 0.15 mg l^?1 1‐naphthaleneacetic acid. The addition of 2.5 mg l^?1 AgNO₃ was very beneficial to shoot regeneration in B. napus and Ag₂S₂O₃ (10 mg l^?1) was even superior to AgNO₃ (2.5 mg l^?1). Explant age, explant type and carbon source also significantly affected shoot regeneration. Four‐day‐old seedlings of cotyledonary explants showed the maximum shoot regeneration frequency and number of shoots per explant. Of the four explants – peduncles, hypocotyls, cotyledons and leaf petioles – cotyledons produced the highest shoot regeneration frequency (56.67 %). Four carbon sources – glucose, maltose, starch and sucrose – were compared for their respective effects on shoot regeneration from cotyledonary explants. Sucrose appeared to be the best carbon source for shoot regeneration with the highest shoot regeneration frequency (76.00 %). Considerable variation in shoot regeneration from cotyledonary explants was observed both between and within Brassica species. The shoot regeneration frequency ranged from 10.00 % for cv. R5 (B. rapa) to 83.61 % for cv. N1 (B. napus). Two B. napus, one B. carinata and one B. juncea cultivars exhibited shoot regeneration frequency higher than 70 %. In terms of the number of shoots produced per explant, B. rapa showed the highest variation, ranging from 5.64 for cv. R3 to 1.33 for cv. R5. Normal plantlets were regenerated from all induced shoots and developed normally. The regenerated plants were fertile and identical with the source plants.     

An evaluation of seed and seedling drought tolerance screening tests in wheat

Summary A series of experiments was performed in order to evaluate the significance of seed germination and seedling growth in osmotic media as screening methods for drought tolerance.Ten spring wheat (Triticum aestivum L. em Thell.) and one durum wheat (Triticum durum Desf.) were tested under controlled environments, using polyethylene glycol-6000 (PEG) solutions as the moisture stress inducing media.Tolerance in the rate of endosperm utilization, under stress, prior to the onset of germination varied among cultivars.Germination rate or injury to germination at various concentrations of PEG differed significantly among cultivars. Cultivar rating with respect to injury to germination changed with stress levels. Injury to germination did not correlate with endosperm utilization rate in PEG or in water.Germinating seedlings were tolerant to extreme desiccation up to the stage of emergence of the first leaf from the coleoptile.Growth of photosynthesizing seedlings was monitored as they were carried through an increasing concentration gradient of PEG solutions, ranging from –5.9 to –11.3 bars of water potential. Cultivars significantly differed in seedling growth tolerance to increasing levels of water stress. Seedling growth tolerance across cultivars was not correlated with their germination responses under srress.It is concluded that tolerance to water stress in growing seedlings can be screened for by using PEG-containing nutrient solutions. It can not be predicted from germination tests in osmotica.Work was done under a US-Israel Binational Science Foundation (BSF) Grant no. 1654/78.Contribution from Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 192-E, 1979 series.     

Production of Primary Triticale from Calluses of Immature Abnormal Hybrid Embryos between Triticum durum and Secale cereale

Plant regeneration was attempted in callus induced from the immature abnormal hybrid embryos between T. durum and S. cereale, using 4-Huorophenoxyacetic acid as a growth regulator. In particular, the relationship of numerical variation in chromosomes between the callus tissues and the regenerated plants was investigated. Cytological observation revealed that there was no distinctive numerical difference between the shoot-forming (SF) and the non-shoot-forming calluses and also between the SF calluses and the regenerated plants. The root-tips of regenerated plants consisted of cells having various chromosome numbers, including the expected 2 n = 3 ×= 21 (genomes, ABR) of which the frequency was 69.8 %. The regenerated plants showed partial fertility, notwithstanding that the hybrid plants were expected to be sterile. Since the frequency of abnormal embryos was about 90 % in this cross, the utilization of abnormal embryos was demonstrated by use of callus culture.     

非洲菊离体叶培养诱导不定芽研究

为探寻非洲菊离体叶片培养的不定芽再生条件,完善非洲菊离体叶片的不定芽再生体系,以不同解离方式（叶自株丛基部撕脱、叶自株丛基部剪切、叶柄剪半）的离体叶为外植体,进行不同激素浓度配比的试验。结果表明,除叶柄剪半的外植体无不定芽再生外,另2种离体叶在叶柄基部均有器官型再生不定芽和器官发生型再生不定芽。自株丛基部撕脱的离体叶于1/2MS+KT 5 mg/L +NAA 0.1 mg/L +蔗糖30 g/L的培养基中,器官型不定芽的直接再生率最高达60%~70%。再生比例为1~2,器官发生型再生率20%~30%,再生比例1~2。器官型不定芽再生主要集中在接种后4~12天的时间段内,而器官发生型不定芽形成主要集中在接种后12~24天的时间段内。若将KT换成BA的不同浓度,则仍以自株丛基部撕脱离体叶的器官型再生率最高达60%~70%,再生比例4~5,器官发生型再生率为50%~60%,再生比例为2~3。试验表明：BA和KT二者同属细胞分裂素类物质,二者浓度上的变化对不定芽再生影响不大,而二者种类不同对不定芽再生差异很大。     

In vitro conservation of tropical plant germplasm — a review

Summary In vitro medium term conservation of tropical plant germplasm is used routinely in many laboratories. Growth reduction is achieved by modifying various parameters, such as temperature, culture medium, gaseous environment. For long term conservation, cryopreservation (i.e. storage in liquid nitrogen, –196°C) is the only current method available. Each successive step of the process requires precise conditions which have to be defined for each material. Cryopreservation protocols have been set up for more than 40 tropical species. Results obtained with various culture systems such as cell suspensions, protoplasts, calluses, meristems and embryos are discussed. The first example of the large scale application of cryopreservation (oil palm somatic embryos) is presented.     

The effects of kinetin on callus characters in alfalfa (Medicago sativa L.)

Summary Three callus initiation media, B2-k, B2, and 7951, were used to study the effects of kinetin on callus initiation, morphology, histology, and regenerability in alfalfa (Medicago sativa L.). The presence of kinetin in callus initiation media retarded callus initiation, but enhanced division and differentiation of callus cells. Calluses induced on kinetin-containing media (B2 and 7951) had many compact cell aggregations, which were considered meristematic regions that might differentiate to plantlets on a regeneration medium. Visually, these calluses were compact and had many nodular structures. In contrast, most calluses induced on a kinetin-free medium were composed of large, individual cells and had friable structures without nodules. After transfer to a hormone-free medium, calluses induced on kinetin-containing media regenerated more frequently than those induced on a kinetin-free medium, but cytokinin (kinetin) autotrophism also occurred. Autotrophism was sexually transmissable and especially affected by the female parent.     

Some evidence for genetically controlled variation in photosynthetic rate of oil palm seedlings

Summary The rate of uptake of ¹⁴CO₂ by oil palm seedlings in relation to light intensity was studied in a complete diallel cross. Analysis showed heritable differences in both light saturated and light limited photosynthesis, and in leaf chlorophyll content. The potential importance of this in a breeding programme is discussed.     

Plant regeneration via organogenesis and somatic embryogenesis in callus cultures derived from mature zygotic embryos of leek (Allium ampeloprasum L.)

Summary A high frequency plant regeneration system via organogenesis and somatic embryogenesis was established with callus cultures derived from mature zygotic embryos of different leek genotypes (Allium ampeloprasum L.). Four different callus types with varying morphogenetic potential were obtained. Relatively high concentrations of the auxin 2,4-dichlorophenoxy-acetic acid reduced callus weight and subsequent shoot regeneration and primordia formation of the callus. Shoot regeneration and primordia formation of the callus decreased after prolonged subculture on media containing 2,4-dichlorophenoxy acetic acid. A callus growth period of six weeks on Murashige and Skoog medium with 0.25–0.5 mg l^-1 2,4-dichlorophenoxy acetic acid showed the highest rate of shoot regeneration after transfer of callus to regeneration medium with 1 mg l^-1 kinetin.Differences between leek genotypes in callus type, callus weight, shoot regeneration and primordia formation were observed. Histological observations showed that plant regeneration took place, both via the pathway of somatic embryogenesis and organogenesis.Abbreviation 2,4-D 2,4-dichlorophenoxy acetic acid - MS Murashige and Skoog (1962) medium     

Stimulation of adventitious bud production in detached sweet potato leaves by high levels of nitrogen supply

Summary Treatment of rooted sweet potato (Ipomoea batatas (L.) Lam.) leaves with high levels of nitrate-nitrogen supply (210 ppm) in sand culture, stimulates the development of adventitious buds and shoots. Up to six adventitious shoots have been produced on a single rooted leaf, grown at 210 ppm NO₃–N. The method may be useful in the production of non-chimeral mutants in mutagenic breeding of sweet potato. It is being used to study physiological factors affecting bud and shoot development as well as tuberisation, as they influence the longevity of the rooted leaf. The method is also being investigated as a technique for the conservation and rapid multiplication of sweet potato genetic resources.     

A procedure for the rapid screening of strawberry seedlings for resistance to Verticilluum wilt

Summary A technique is described which promises to be of great assistance in breeding for resistance to verticillium wilt in strawberries.Strawberry seeds were sown on moist terylene wool, covered with black polyethylene beads and suspended over aerated liquid nutrient solution. Good germination of the seeds occurred, and the seedlings developed rapidly reaching the third-leaf stage with a well-established root system after 45 days. At this stage, the seedlings were inoculated with pathogenic isolates of Verticillium dahliae Kleb, by immersing the root systems in a conidial suspension (100000/ml) for 1 h, then replacing the seedlings in the liquid nutrient solution. Wilt symptoms, i.e. leaf wilting and necrosis, were first observed on the most susceptible seedlings 21 days after inoculation. All seedlings were re-inoculated 35 days after the initial inoculation, and those which were visually healthy 50 days later were graded as tolerant and subjected to further tests.     

播期和密度对棉花叶柄和根系硝态氮含量的影响

为探讨大田棉花氮代谢随播期和密度的变化规律,选用华棉3109(G.hirsutum L.)于2014年在华中农业大学试验农场,采用裂区设计:播期(月-日)(S1,05-30;S2,06-14)为主区,密度(株·m-2)(D1,7.5;D2,9.0;D3,10.5)为副区,研究了硝态氮含量在主茎叶柄和根系的分布特点。结果表明:1)随生育进程推进,叶柄和根系硝态氮含量先升高后降低,初花期最高。2)主茎叶柄硝态氮含量随叶位变化,蕾期、初花期由上而下逐渐降低,第1叶最高;盛花期逐渐增高,第1叶和第4叶最高;不同生育时期棉花叶柄硝态氮含量在叶位间的下降幅度随播期推迟而降低,随密度增加先升高后降低。3)播期和密度对不同生育时期棉花叶柄和根系硝态氮平均含量的交互作用均显著,但播期和密度主效应影响不同:见花施肥前,随推迟播期,棉花叶柄硝态氮平均含量显著降低了42.9%,根系硝态氮平均含量显著升高了12.1%,增加密度对叶柄和根系硝态氮平均含量无显著影响。见花施肥后,随播期的推迟,叶柄硝态氮平均含量无显著性变化,初花期平均为5.05 mg·g-1,盛花期平均为2.62 mg·g-1;而根系硝态氮平均含量,初花期S1S2,盛花期S1S2;随密度增加,D1,D2与D3初花期叶柄和根系硝态氮平均含量均显著降低;盛花期叶柄硝态氮平均含量呈先升高后降低趋势变化,而根系硝态氮平均含量则与初花期相反,呈显著递增趋势。综上所述,晚播高密条件下,见花一次施肥后,推迟播期不改变棉花地上部叶柄硝态氮平均含量水平,适度增加密度有利于棉花叶柄维持较高的硝态氮含量,有利于为叶片氮代谢提供充足的底物。     

Reproductive capacity of F1 hybrids from Rosa rugosa and Chinensis cultivars

Summary Reciprocal crosses were carried out for 6 combinations of 4 R. rugosa with 3 R. chinensis cultivars. The parental combinations were evaluated for successful pollinations, seed germination and flowering and seed setting seedlings in populations of 3 year old plants. Seeds and viable seedlings were obtained from each of the 12 seedling populations. Only 3 populations produced female fertile offssprings. The average of seed setting seedlings was 1.5%. The percentage of flowering seedlings was significantly reduced in the offspring from certain parents. The success of cross pollination, seed germination and the ability to initiate flowers were influenced by cytoplasmic factors.The differences in fertility between the present hybrids and the only other rugosa x chinensis hybrid described, R. calocarpa (Andrê) Willmott, are discussed.Contribution No. 73/2.     

Plant regeneration in sweet potato (Ipomoea batatas L., Convolvulaceae)

The application of new techniques for improvement of sweet potato crops, particularly including the exploitation of somaclonal variation, gene transfer by genetic transformation and somatic hybridization, requires the control of plant regeneration from tissue cultures. Shoots can easily be regenerated from explants of stems, petioles, leaves and roots, while callus cultures do not produce any shoots. The potential of somatic embryogenesis and plant regeneration via embryogenesis was evaluated for 10 cultivars of sweet potato. Protocols for plant regeneration from cultured protoplasts have also been developed. Since mesophyll was resistant to enzyme digestion, fragments of stems and petioles, callus and cell suspensions were used as source of protoplasts of sweet potato. Series of transfers of protoplast-derived calluses, particularly those which had been obtained from in vitro plants, to media containing a high level of zeatin resulted in successful formation of shoots in only two sweet potato cultivars. In addition, the embryogenic potential was irreversibly lost through protoplast culture, since protoplasts isolated from embryogenic cell suspensions developed into non-embryogenic callus. Consequently, an alternative protocol is being successfully developed to improve plant regeneration from cultured protoplasts of sweet potato, involving first root formation from which shoots can then be regenerated. Preliminary evaluation in field conditions in Gabon revealed that plants regenerated from cultured protoplasts exhibited a great genetic variability in their growth and tuber formation in particular. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of tetraploid plants from an interspecific hybrid between mungbean (Vigna radiata) and rice bean (Vigna umbellata)

Mungbean (Vigna radiata) and rice bean (V. umbellata) (both species 2n = 2x = 22) have desirable traits that complement each other. In this study, we rescued embryos from a cross between mungbean cv. “Kamphaeng Saen 2” and rice bean cv. “Miyazaki” and resolved the hybrid sterility problem by colchicine treatment. The interspecific hybrids were obtained when Kamphaeng Saen 2 was used as the female parent. Four out of 80 immature seeds at 12 days old were able to germinate on an MS medium supplemented with 1 mg L^?1 IAA, 0.2 mg L^?1 kinetin, and 500 mg L^?1 casein hydrolysate. Forty random amplified polymorphic DNA (RAPD) primers were screened for polymorphism among the parents, and two specific primers were finally chosen for testing of hybridity. Using the two primers, all putative F₁ hybrids were confirmed as the interspecific hybrids. To observe their fertility, some of the hybrid seedlings were transplanted. The hybrid produced flowers profusely but failed to set pods. To overcome the sterility, plants were induced to become tetraploid by colchicine treatment in vitro. The ploidy level of the regenerated seedlings was confirmed from leaf DNA using a flow cytometer. Three out of 20 hybrid seedlings (15%) were successfully induced from diploid to tetraploid by a colchicine concentration of 2 g L^?1. The tetraploid hybrids were able to produce flowers and set pods normally.     

籼、粳、糯稻成熟种胚体细胞培养力的差异

在相同的培养条件下,比较了不同类型水稻成熟种胚体细胞培养力的差异。结果,糯稻的愈伤组织诱导率和生长速度高于粳稻和籼稻。就愈伤组织质地和色泽而论,籼稻趋向于紧密型、淡黄色,粳稻趋向于松散型、鲜淡黄色,糯稻趋向于粘质状、淡黄色。而籼稻的愈伤组织绿苗分化率、绿苗+绿芽分化率和体细胞培养力高于粳稻和糯稻。这些结果为利用种胚培养进行水稻品种改良提供了一定的理论依据。