离体植物花芽和花器官的发育研究进展

在近二十几年,尤其是近十年由于离体培养技术的完善和进一步向精确的层次的发展,已使得许多种植物花序、花芽和单独花器官以及部分的花器官在体外成功地进行了培养和尝试,并且对花芽及花器官的离体发育有了更深入的认识。不同植物的花发育需要不同的植物生长调节剂以及它们的不同配比,并且不同植物在其花发育所需要的营养因子也存在着相当大的差异性。这种差异性又随植物器官及花发育的不同阶段而受到加大或缩小。通过对正常花及突变体花进行的离体培养实验研究已经对一些花器官发生过程中的调节程序有了新的了解。利用离体培养技术,包括刚发展起来的薄层细胞培养技术在阐明花发育的机理、形态建成的分子机制以及成花梯度的物质基础等问题上具有广阔的潜力。     

Experimental control of floral reversion in isolated shoot apices of the long-day plant Silene coeli-rosa

The fate of shoot meristems of the long day (LD) plant Silene coeli-rosa in culture was examined (complete, reverted or arrested flowers) to establish whether these different patterns were related to a particular stage of morphogenesis and the extent to which the fate of the pattern was regulated by either added plant growth regulators (PGRs) or changing the carbohydrate source in the medium. In particular, the frequency of reversion was measured to test the stability of the determined state for each whorl. The plants were given various inductive treatments (4–7 LD, 7 LD + 1 to 3 SD) and the apices were explained onto Murashige-Skoog medium supplemented with 3% sucrose (controls) ± IAA, ± kinetin, ± GA₃ or onto the basal medium containing 1 or 3% sucrose, glucose or fructose or 7% sucrose. The apices were examined 12 weeks later. When the data were pooled from all inductive treatments, IAA resulted in more reversions, GA₃ caused more arrested flowers while kinetin hardly affected the pattern of meristem fate compared with the controls. However, each PGR treatment did not perturb the pattern of organ formation for those apices that formed either arrested or complete flowers. The time for determination (days) of the earlier formed whorls (determination times for the controls in brackets): sepals (2), stamens 1–5 (3) and petals (3), was shortened by about a day in all PGR treatments whereas the corresponding times for the later formed whorls: stamens 6–10 (4) and carpels (4), were either lengthened to 5 days or unaffected. The response of the apices to the various sugars was simply a reflection of concentration. Hence, more complete flowers formed at 7 or 3% and more flowers were arrested at 1 % regardless of the sugar moiety. However, the frequency of reversion was similar on each of the media. Pooling all data from all treatments enabled a statistical analysis of the pattern of reversion and the pattern of arrest. Reversion was more common from apices which exhibited the later-formed whorls (stamens 6–10 and carpels) than from the earlier whorls. Moreover, the stronger the inductive treatment the less frequent was reversion. The most common stage of arrest was at the stamen 6–10 whorl and this was particularly so for the GA₃ treatment. The data indicated that reversion could occur from any whorl, which suggests that determination of each whorl is independent of the next. This conclusion is underlined by the more frequent occurrence of reversion from the carpel whorl. However, the longer the inductive treatment the less likelihood of reversion; this suggests that in Silene, the floral stimulus is required continuously to stabilise the determined state of each whorl and to ensure smooth completion of floral morphogenesis.     

In vitro plant regeneration from leaf callus of Grindelia robusta Nutt

Abstract

A regeneration protocol from leaf explants of Grindelia robusta Nutt. was developed. The combination of 0.5 mg l^?1 IBA plus 0.5 mg l^?1 or 1 mg l^?1 BA added to Murashige-Skoog (MS) medium resulted in the best callus induction frequency; the combination of 0.4 or 0.9 mg l^?1 BA plus 1.2 mg l^?1 GA₃ resulted in the best shoot regeneration. Rooting was successful on MS medium supplemented with 0.5 mg l^?1 IBA. Hardening of G. robusta plants was accomplished in 30 days with 85% survival rate.     

Mutants involved in floral and plant development in Petunia

The Petunia system is described together with its possible use for molecular research on floral development. Developmental mutants are described in some detail. The most interesting mutants are Blind and Green petals. In both the appearance of only the petals has changed, in Blind towards antheroid tissue, in Green petals towards sepals. Transposon tagging as an approach to isolate developmental genes in Petunia is also discussed.     

In vitro shoot regeneration from cotyledons of immature ovules ofImpatiens platypetala Lindl.

Summary An efficient and reproducible protocol has been developed for in vitro shoot regeneration from cotyledonary explants derived by germinating immature ovules ofImpatiens platypetala Lindl. ‘TR6-27-2’. Cotyledonary explants were cultured on a modified Murashige and Skoog (MS) agar-solidified medium containing 7.5g · liter^−1 sucrose, 22.2µ M N⁶-benzyladenine (BA), and 0.54µM α-naphthaleneacetic acid (NAA). The induction of organogenic tissues occurred after 6 to 8 wk in culture. Exogenous auxin and cytokinin were essential for the induction of organogenic tissues and survival of explants, and BA was most effective for the induction of organogenic tissues, compared with other cytokinins tested. The addition of glutamine (500 mg · liter^−1) was also important for growth of organogenic tissues after induction and for reducing explant death during culture. The induction of organogenic tissue was also influenced by the type of cotyledon cultured and the age of the donor seedlings. On average, eight shoots per explant were induced from organogenic tissues larger than 0.5 cm in diameter 6 to 8 wk after transfer to a modified MS agar-solidified medium without NAA and BA reduced to 4.44µM. Shoots longer than 0.5 cm in length were successfully rooted 2 to 4 wk after transfer to a basal MS medium containing 30g · liter^−1 sucrose.     

花生叶片离体培养花器官分化及其开花结果研究

以花生幼叶为外植体进行离体培养,研究BA浓度对花器官分化的影响并进一步观察试管内花器官的发育.结果表明:经MSB 1mg/LBA 0.5mg/LKIN 2mg/LIAA培养基诱导的愈伤组织,转接到附加1～3mg/LBA的MSB培养基上培养,均能直接诱导分化花器官,但2mg/LBA的诱导效率最高达21.13%;诱导分化的花器官转接到MSB培养基继续培养,部分花器官可以在试管内开花、受精、成针、结实.试验实现了以花生幼叶为外植体,在试管内完成诱导花芽、开花、受精、形成果针、子房膨大,直至形成荚果等过程,为离体条件下研究花生花器官分化、荚果及种子发育提供了技术体系和材料.     

不同辣椒材料离体再生及其影响因素的研究

以8个辣椒(Capsicum annuumL.)纯系为材料,对不同材料、外植体种类和激素组合等因素对辣椒植株离体再生的影响进行了研究。结果表明,较高的6-BA/IAA值有利于辣椒外植体的分化再生,而6-BA/IAA值较低则适合于再生芽的伸长;不同辣椒材料的再生能力差别较大;辣椒带柄子叶再生能力比下胚轴强,是较好的外植体材料;12~16 d苗龄的外植体分化频率较高;在供试的8个辣椒材料中‘2096’、‘B4’和‘B7’的再生能力较强。高频率的不定芽分化培养基为MB(MS无机成分 B5有机成分) 0.8 mg/L IAA 5.0 mg/L 6-BA 4.0 mg/L Ag-NO3;不定芽伸长的培养基为MB 0.8 mg/L IAA 2.0 mg/L 6-BA 2.0 mg/L GA3 4.0 mg/L AgNO3;高效生根诱导培养基为MB 0.2 mg/L IAA 0.1 mg/L NAA。     

In vitro propagation of cauliflower,Brassica oleracea var. botrytis for hybrid seed production

Methods for obtaining heterotic F₁ and maintaining purebred lines for breeding of Brassica oleracea are limited by absence of male sterile lines and occurrence of inbreeding depression, respectively. The use of vegetative (stem, petiole, leaf, leaf rib) and floral (peduncle, pedicel, flower bud, curd) explants of cauliflower to regenerate purebred lines for crossing were examined. Of four growth regulator treatments and explant types used, best results were obtained with curd explants on MS medium with 6-benzyladenine (cytokinin) and gibberellic acid. Although 6-benzyladenine alone promoted formation of shoots in floral explants, both 6-benzyladenine and α-napthaleneacetic acid were required for vegetative explants. Use of α-napthaleneacetic acid, however, often increased callus formation. These culture techniques to maintain purebred regenerated plants will complement newly-derived nuclear-based male sterile lines obtained by the introduction of antisense copies of the gene BcpI, which is required for pollen fertility. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro development of Cryptosporidium parvum in serum-free media

AIM: To compare commercially available serum-free media with common, standard, growth medium for their ability to support growth of Cryptosporidium parvum in HCT-8 cell cultures. METHODS AND RESULTS: Twelve serum-free media formulations with or without additional supplements were tested against a standard growth medium containing 2% FBS in HCT-8 cell cultures. After a 48-h incubation period, the level of parasite development was determined by ELISA. The extent of development in the serum-free media was determined as a percentage of infections compared with those obtained using a standard growth medium. CONCLUSIONS: Several of the serum-free media formulations, which included MDCK, UltraMDCK, PC-1, UltraCHO and UltraCulture, compared favourably with a traditional, standard growth medium. Moreover, increasing FBS concentrations to 10% actually resulted in an overall decrease in development in many cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Several serum-free medium formulations are available which allow development of C. parvumin vitro at levels comparable with standard media employing FBS. These serum-free media are particularly useful for applications, which may require a more defined medium without the presence of FBS. Moreover, the elimination of FBS as a variable allows investigators the ability to more closely regulate their experimental systems when growing C. parvum in cell cultures.     

Vaxiations in floral development in Peperomia (Piperaceae) and their taxonomic implications

The floral development of two species of Peperomia, Peperomia reflexa A. Dietr. (P. tetraphylla (G. Font.) Hook. et Am.) and P. serpens C. DC., is described. The initiation order is in an acropetal succession and resembles that in P. metallica L. Lind. et Rod., P.pellucida (L.) Kunth and P. rubella Hook., although many more bracts are produced in these two species than in P. metallica. The arrangement of bracts and floral primordia is orthostichous in P. reflexa, but parastichous in P. serpens. The floral apices in P. reflexa are transversely ellipsoidal protuberances at first, then become saddle-shaped when they begin to produce simultaneously staminal primordia. They are similar to those of P. metallica, P. pellucida and P. rubella. However, their initiation of floral primordia is much delayed compared to the size of the bracts. The triangular or transvcrsely cuneate ridges then become L-shaped in P. serpens, this shape is related to the parastichous phyllotaxy of the bracts. So, the staminal primordia are successively initiated and develop at different rates. Therefore, they are not always the same size. The staminal primordia are initiated above the level of the floral apex in P. reflexa and P. serpens but below it in P. metallica, P. pellucida and P. rubella. The abaxial position of the carpel primordium on the apex and the closure of the ovary in P. reflexa and P. serpens are also similar to those in P. metallica, P. pellucida and P. rubella. The shape of the upper part of the ovary, stigmas and indumentum vary between the species. In P. reflexa, the upper part of the ovary becomes ovoid and acclivous, or leaning acropetally, to the axis of inflorescence. In P. serpens, it becomes helmet-shaped. The flowers of P.serpens are surrounded by the outgrowth of the axis of the inflorescence. The ontogenetical features of ovaries in Peperomia indicate that the fruit characters are useful in the taxonomy of the genus.     

In vitro pollination of angiosperm ovules with gymnosperm pollen grains

Summary Pollen grains of the gymnosperm species Ephedra distachya and Pinus wallichiana germinated abundantly on the in vitro cultured placentae of the angiosperm species Nicotiana tabacum, Melandrium album and Allium moly. Some P. wallichiana pollen tubes entered the micropyles of M. album. Embryological observations of cross-pollinated M. album ovules 2 or 3 d after cross-pollination revealed the presence of pollen tube remnants within the embryo sacs. Karyological disturbances in the two- and three-celled proembryos and endosperm nuclei indicated their probable hybrid origin. In some crosses, gynogenetic haploid proembryos were also noticed.     

Number of floral organs in Circaeaster agrestis (Circaeasteraceae) and possible homeosis among floral organs

98.9% of 5092 flowers from 1041 individuals of Circaeaster agrestis have five floral organs, the formula is P₃A₁G₁ (73.13%), P₂A₂G₁ (25.59%), and P₂A₁G₂ (0.22%). Only 0.4% of the flowers have six floral organs and the formula is P₃A₁G₂ (20 flowers) or P₃A₂G₁ (one flower). All these flowers have one vascular bundle in the pedicel and were considered to be normal ones. There are 33 flowers (0.65%) with six or more floral organs and two vascular bundles in the pedicel and we found traces of fusion of different degree of two flowers into one. These flowers were considered as abnormal. Therefore the normal number of floral organs of C. agrestis is five and occasionally six, and the floral formulas are P₃A₁G₁ or P₂A₂G₁, sometimes P₂A₁G₂, and occasionally P₃A₁G₂ or P₃A₂G₁. A tepal in P₃A₁G₁ may be replaced by a stamen in P₂A₂G₁ or by a carpel in P₂A₁G₂ or in reverse. A carpel in P₃A₁G₂ may be replaced by a stamen in P₃A₂G₁ or in reverse. We hypothesize that there are two possibilities for the number of the floral organs to be five (six), the result of reduction from P₃A₂G₂, or there exists homeosis among floral organs.     

In vitro phyllomorphic regeneration of shoot buds and shoots in Picea abies

Needles (10–15 mm) of frost-hardened 20–22-week-old (physiological age equivalent to 1 year) plants of Picea abies L. excised just after flushing, were induced to form adventitious shoot buds and shoots on media supplemented with BAP (6-benzylaminopurine) and NAA (1-napthaleneacetic acid). The addition of nanomolar concentrations (0.5–50) of NAA combined with 1–10 μM BAP considerably stimulated formation of pseudobulbils on the basal to mid-part of the needle axis, as well as their subsequent development into shoot buds and shoots. On a medium containing 10 μM BAP, pseudobulbils that formed at the needle base did not develop further, but became necrotic and died with the omission of NAA. With 5 μM BAP + 50 nM NAA the initial phase of development was slow, but later showed good response and up to 22% of the needles produced shoot buds. Two to three shoots per needle could be excised and subcultured individually onto fresh media. It is concluded that the level of endogenous auxin decreases progressively from the needle's base to its tip, so that that concentration of exogenous auxin (50 nM NAA) which promotes pseudobulbil and shoot-bud formation part-way along the needle axis, simultaneously inhibits their induction at the needle base.     

In vitro flowering of Dendrobium candidum

Dendrobium candidum, a wild orchid species from China, normally requires three to four years of cultivation before it can produce flowers. The effects of plant hormones and polyamines on flower initiation of this species in tissue culture were investigated. The addition of spermidine, or BA, or the combination of NAA and BA to the culture medium can induce protocorms or shoots to flower within three to six months with a frequency of 31.6% -45.8%. The flowering frequency can be further increased to 82.8 % on the average by pre-treatment of protocorms in an ABA-containing medium followed by transfer onto MS medium with BA. The induction of precocious flowering de-pends on the developmental stage of the experimental materials (protocorms, shoots and plantlets) used , and usually occurs only when root formation is inhibited.     

In vitro establishment of a cycloclonal chain from nodal segments and apical buds of adult hazel (Corylus avellana L.)

Apical buds (0.5 cm) and nodal shoot segments (1.5 cm) excised from: A) field-grown branches, B) newly developed shoots from the forced outgrowth of axillary buds on A branches, C) newly developed shoots from the forced outgrowth of axillary buds on A branches submitted to cold storage were used as primary explants. Results indicate that three months cold storage greatly increases morphogenic capacity and reduces contamination and oxidation of tissues. Consequently, a multiplying chain could be easily established by culturing the tissues on a modified Murashige & Skoog (1962) medium plus 6-benzyl-aminopurine 5 mg l^-1, indole-3-acetic acid 0.01 mg l^-1 and gibberellic acid 0.1 mg l^-1. During the initiation and proliferation phases, both the proliferation and the elongation rate were significantly increased when a double-phase culture system (Viseur 1987) was used, giving rise to a higher microplant production than the one obtained using previously described methods. Plant regeneration was achieved by immersing the single microshoot's basal end in an IBA (0.1–1 mg ml^-1) solution for 10 s followed by a 20-day culture on a 1/2 MS2 medium.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - MS1 and MS2 modified Murashige & Skoog media - NAA 2-maphtaleneacetic acid     

枸杞髓组织离体培养及高频率植株再生的研究

枸杞髓组织在４种ＭＳ培养基上都能诱导出愈伤组织,诱导率５３．７％～１００％。在培养基ＭＳ＋６－ＢＡ０．１ｍｇ／Ｌ＋ＮＡＡ０．５ｍｇ／Ｌ获得的愈伤组织,呈颗粒状,分散性能好,胚性细胞多．将其转移到ＭＳ＋６－ＢＡ０．５ｍｍ／Ｌ＋ＮＡＡ０．０１ｍｇ／Ｌ的分化培养基上获得大量绿色小芽,小芽在ＭＳ＋６－ＢＡ０．２ｍｇ／Ｌ的培养基上得到快速繁殖,繁殖系数５０～１５０株／芽·月。丛生芽在ＭＳ＋ＮＡＡ０．２ｔｍｇ／Ｌ的培养基上形成完整植株     

In vitro formation of axillary buds by immature shoots of Ponderosa pine

Axillary buds were induced from immature shoot explants taken from terminal buds of branches from 29- and 34-year old ponderosa pines (Pinus ponderosa Dougl ex Laws). The effect of collection time, position on the donor tree from which the explants were taken, and plant growth regulators on axillary bud formation was investigated. Explants from branches taken in late October formed axillary buds, whereas explants from branches collected in February 1988 produced a large amount of callus. The ability to form axillary buds was significantly greater for explants from the upper crown than from the lower portion of the tree. Explant elongation occurred and basal needle primordia swelled on Murashige & Skoog media (MS) containing 2.2 M 6-benzyladenine (BA) and 5.4 M naphthalenacetic acid. When transferred to a MS medium containing 4.4 M BA, 59% of explants formed axillary buds.     

Morphology and development of floral shoots and organs in certain Zannichelliaeeae

Development of reproductive shoots and associated organs in Vleisia aschersoniana, Althenia filiformis, Lepilaena bilocularis and L. cylindrocarpa (Zannichelliaeeae, sensu Dumortier) has been examined using an epi-illumination technique to provide photographic documentation. Floral shoots share a similar basic developmental pattern. The vegetative shoot is terminated by a unisexual flower, but growth is continued from the axils of leaves immediately below so that fairly regular sympodia develop. Vleisia is most variable in the expression of this pattern. The flowers are simple, consisting of a single stamen or three carpels (one in Vleisia) and show marked similarity in development. Short scale-like appendages, reminiscent of a perianth, develop at the base of the stamen in Althenia and Lepilaena. An outgrowth at the tip of the connective in Lepilaena bilocularis and two at its middle part in Vleisia are initiated at late stages of stamen development. Carpels are subtended by membranous tepal-like appendages that are initiated at the same time as the carpel primordia. Each carpel primordium becomes peltate and develops a bitegmic ovule on the adaxial portion of the carpel wall which in turn overgrows the ovule and ultimately forms a long thin style with either a funnel-shaped (Vleisia, L. cylindrocarpa) , peltate (L. bilocularis) or feathery (Althenia) stigma. Relationships with other Alismatales are discussed.     

In vitro development of plants from microspores of rice

Summary Rice (Oryza sativa L., 2n=24) anthers containing microspores in the early-uninucleate to first-mitosis stages were induced successfully to develop into plants in vitro through an intermediary step of callus formation. Callus initiation occurred with highest frequency in anthers containing mid-uninucleate microspores. The callus derived from different stages of microspore development differed in the potential to differentiate into plants. The plants regenerated from pollen callus were predominantly haploid or diploid; polyploid and aneuploid plants were relatively infrequent. The first division of the uninucleate microspores was asymmetrical, resulting in the formation of large vegetative and small generative nuclei. The vegetative nucleus divided repeatedly and assumed the major role in the formation of callus, whereas the generative nucleus degenerated rapidly. Simultaneous division of the two nuclei was observed in a few pollen grains. Nuclear fusion during the very initial stages of pollen development was postulated to account for the occurrence of the diploid and polyploid plants. This work was supported by the National Science Council, Republic of China.     

黄瓜花芽启始分化的形态解剖研究

苗龄 6d的黄瓜幼苗 ,在第一节位叶腋处花芽原基开始启动分化。花芽分化时间早、速度快、节位低、同步性好。诱导黄瓜开花的因素可能不是光和夜低温 ,其开花特性类似于自主开花植物