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1.
Microspore cryopreservation is a potentially powerful method for long-term storage of germplasm for in vitro embryo production in plant species. In this study, several factors influencing embryo production following the ultra-low temperature (–196 °C in liquid nitrogen) storage of isolated microspores of rapeseed (Brassica napus L.) were investigated. Microspores were prepared in cryogenic vials and subjected to various cooling treatments before immersion in liquid nitrogen for varying periods. Efficiency of microspore cryopreservation was reflected by in vitro embryo production from frozen microspores. Of all the cooling treatments, microspores treated with a cooling rate of 0.25% °C/min and a cooling terminal temperature of –35 °C before immersion in liquid nitrogen produced the highest embryo yields (18% and 40% of unfrozen controls in two genotypes, respectively). Fast thawing in a 35 °C water bath was necessary to recover a high number of embryos from microspore samples being frozen at a higher cooling rate, while thawing speed did not affect samples after freezing at a slower cooling rate. The storage density of cryopreserved microspores affected embryo production. Storage at the normal culture density (8×104 microspores/ml) was less efficient for embryo production than at high densities (4×106 microspores/ml and 1.6×107 microspores/ml), although no significant difference was found between the high densities. Evaluation of plant lines derived from frozen microspores indicated no variation in isozyme pattern and no enhanced cold tolerance of these lines. Isolated microspores of B. napus could be stored for extended period for in vitro embryo production. 相似文献
2.
J. J. Rybczynski R. L. Simonson P. S. Baenziger 《In vitro cellular & developmental biology. Plant》1991,27(4):168-174
Summary In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus.
Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther
culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis.
Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation
of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation
of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with
suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were
cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per
responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos
could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration
medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential
to use this information to enhance plant production is discussed. 相似文献
3.
The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability
of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed
no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine,
myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of
the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures
with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid
clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in
the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were
asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic
fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores,
indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect
of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation
of multinuclear structures.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
4.
Embryogenesis and plant regeneration of hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) through isolated microspore culture 总被引:1,自引:0,他引:1
We report high frequencies of embryo production and plant regeneration through isolated microspore culture of hot pepper (Capsicum annuum L.). Microspores cultured in modified NLN medium (NLNS) divided and developed to embryos. Globular and heart-shaped embryos
were observed from 3 weeks after the beginning of culture, and many embryos reached the cotyledonary stage after 4 weeks of
culture. These cotyledonary embryos developed to plantlets after transfer to solid B5 basal medium. We also optimized conditions
for embryo production by varying the pretreatment media, the carbon sources, and culture densities. Heat shock treatment in
sucrose-starvation medium was more effective than in B5 medium. Direct comparisons of sucrose and maltose as carbon sources
clearly demonstrated the superiority of sucrose compared to maltose, with the highest frequency of embryo production being
obtained in 9% (w/v) sucrose. Microspore plating density was critical for efficient embryonic induction and development, with
an optimal plating density of 8 × 104–10 × 104/ml. Under our optimized culture conditions, we obtained over 54 embryos, and an average of 5.5 cotyledonary embryos when
10 × 104 microspores were grown on an individual plate. 相似文献
5.
Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18). 相似文献
6.
Regeneration of fertile green plants from isolated oat microspores is reported for the first time. Factors critical for microspore
growth and regeneration include cold pre-treatment, pH of culture medium and the use of conditioned culture medium. It was
found that cold pre-treatment at 4°C in the dark for a minimum of 6 weeks was necessary to consistently achieve microspore
growth into multicellular structures (MCS). Longer pre-treatments of up to 9 weeks were tested and found to be positively
correlated with the number of MCS produced. Microspore culture medium with pH 8.0 produced significantly more MCS larger than
eight cells in size than media with pH 5.8. The use of medium conditioned by actively growing barley microspores significantly
increased the numbers of MCS larger than eight cells in size compared to non-conditioned media. Plants were regenerated only
from cultures using conditioned medium. A total of 2 green plants and 15 albinos were regenerated. Of the green plants, one
had the haploid chromosome complement (n = 3x = 21) and the other had the parental hexaploid chromosome complement (2n = 6x = 42) which may be due to spontaneous chromosome doubling. The hexaploid plant set seed naturally and the haploid plant set
seed after its chromosome complement was doubled with colchicine. 相似文献
7.
The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological
examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers
were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding
to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to
embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and
initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten
or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty
days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot,
regenerating plantlets. 相似文献
8.
In cabbage (Brassica oleracea var. capitata), a thermal shock treatment of 24 h at 35 °C at the start of the culture period resulted in higher embryos per 100 anthers (30.0) compared to a treatment of 48 h. Similarly , a chilling treatment of 24 h at 4 °C resulted in a higher embryo yield (6.0) per 100 anthers compared to a treatment of 48 h. However, the embryo yields were significantly higher (p> 0.01) in thermal shock than chilling treatments in all experiments. Treatments of 6 days at either 35 °C or 4 °C gave no embryos. The most responsive cultivar was the F1 hybrid , Hercules, in all experiments. Although anther culture was successful in the other genotypes, the open pollinated ones, the highest number of embryo yields per 100 anthers was obtained in the hybrid. High temperature treatment before culture had a beneficial effect on the embryo yields. The responsiveness of anthers to addition of increasing concentration of silver nitrate (AgN03) (the ethylene inhibitor) to the culture medium, showed a progressive increase in the embryo yields in all the genotypes. Since embryos were also formed in the absence of silver nitrate, probably, due to a greater genotype × medium interaction, it is noted that the presence of silver nitrate in the medium may not be essential for cabbage anther culture as reported earlier. The findings of this study may be recommended for large production of cabbage embryos in culture. 相似文献
9.
The present study was undertaken to establish a culture system for ovules excised at the zygote stage in Lilium spp. Ovules of Lilium × `Connecticut King' and L. × `Enchantment' were excised together with placental tissue 3, 5, and 10 days after pollination (DAP) and cultured on B5 medium and half-strength B5 medium containing sucrose at different concentrations. In vitro embryo development in ovules cultured at 3 DAP was influenced by the basal media and the sucrose concentration. The half-strength B5 medium with 9% sucrose was the best condition, but only a few ovules isolated from placental tissue developed into seedlings. Application of embryo culture, in which embryos were excised from ovules after 14 weeks of ovule-with-plancetal-tissue culture, greatly improved the production of seedlings. The present study indicates that a two-step culture procedure, ovule-with-placental-tissue culture and embryo culture, make it possible to produce seedlings from ovules just after fertilization. 相似文献
10.
Enhanced post-germinative growth of encapsulated somatic embryos of Siberian ginseng by carbohydrate addition to the encapsulation matrix 总被引:5,自引:0,他引:5
Höfer M 《Plant cell reports》2004,23(6):365-370
Based on a protocol for microspore culture in apple (Malus domestica Borkh.), the embryo induction phase has been improved with regard to pretreatment of microspores for initiation of microspore embryogenesis, the concentration of carbon source in the induction medium and the microspore density in the suspension. Furthermore, the effect of the genotype was studied. To determine the efficiency of in vitro androgenesis, both methods, via anther and microspore culture, were investigated using the same bud material. A comparison of the efficiency of embryo induction in anther and microspore cultures showed that microspore culture resulted in an increase up to 10 times, depending on the genotype. The regeneration route in microspore culture is similar to that of androgenic embryos via anther culture and showed adventitious shoot formation in most cases after a long period of secondary embryogenesis.Communicated by H. Lörz 相似文献
11.
A. Touraev A. Indrianto I. Wratschko O. Vicente E. Heberle-Bors 《Sexual plant reproduction》1996,9(4):209-215
We have established an efficient method to induce embryo formation from isolated wheat (Triticum aestivum L.) microspores. Culture of excised anthers under starvation and heat shock conditions induced the formation of embryogenic microspores at high frequency in nine Austrian winter wheat genotypes, including cultivars that had been considered as recalcitrant in anther culture. Percoll gradient centrifugation of the mechanically isolated microspores allowed us to obtain homogeneous populations of embryogenic microspores in all genotypes which, after transfer to a rich medium containing immature ovaries for conditioning, divided and produced globular embryos. Thousands of embryos were produced in one petri dish. Many of these embryos developed into plantlets after transfer to a solid medium without ovaries. 相似文献
12.
Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations
SEM
Scanning electron microscopy
-
TEM
Transmission electron microscopy 相似文献
13.
The 27 lemon cultivars analysed could be considered slightly or moderately polyembryonic, with 25 to 43% of seeds being polyembryonic
and from 1.3 to 1.6 embryos per seed. On this basis, it is necessary to rescue zygotic embryos at an immature stage. Rescue
and in vitro embryo development have been studied in two Citrus limon polyembryonic cultivars. Sucrose (50 and 70 g/l) was combined with Murashige and Skoog and Gamborg’s B5 media and tested
for optimal growth response. An important effect of genotype was observed: embryos from cultivar ‘Eureka’ had greater survival,
germination percentage, and radical development. While the sucrose concentration in the medium did not have an effect on germination,
the medium affected the embryo survival and root development of the seedlings, Gamborg’s B5 medium giving the best results.
The ability to form plants in vitro was affected by an increase of embryo developmental stage. The germination and seedling
height were greater with embryos of seeds collected 135–150 days after anthesis. 相似文献
14.
Microspores of several genotypes of Brassica campestris ssp. parachinensis have been cultured in vitro and induced to undergo embryogenesis and plant formation. Conditions favourable for embryogenesis in this species include a bud size of 2–2.9 mm, NLN-13 culture medium (Nitsch and Nitsch 1967; Lichter 1981, 1982; Swanson 1990), and an induction through exposure to 32°C for a period of 48 h. Longer periods of an elevated temperature for induction of embryogenesis resulted in embryo abortion at early developmental stages. With the protocol developed here, microspores of 60–80% of donor plants could be induced to produce embryos, although embryo yields were low, i.e. 2–5 embryos per 10 buds. Some genotypes responded to culture conditions with high numbers of embryo formation (100–150 embryos per 10 buds) but most of these subsequently failed to mature. The pattern of cell division and morphological changes of the microspores in culture were studied using various microscopic techniques. 相似文献
15.
Somatic embryogenesis and plant regeneration of turf-type bermudagrass: Effect of 6-benzyladenine in callus induction medium 总被引:17,自引:0,他引:17
In order to optimize tissue culture conditions for bermudagrass, an important warm-season turfgrass species, tissue culture
responses of young inflorescences of a hybrid bermudagrass cultivar `Tifgreen' (Cynodon dactylon×Cynodon transvaalensis) and a common bermudagrass cultivar `Savannah' (Cynodon dactylon) were investigated. When cultured on Murashige and Skoog medium with 4.52 to 13.57 μM (1–3 mg l-1) 2,4-D, young inflorescence segments yielded non-embryogenic calli which were unorganized and had loosely associated, long
tubular cells on the surface. However, inclusion of 6-benzyladenine (BA) in callus induction medium at a level of 0.044 μM
(0.01 mg l-1) induced formation of a compact, nodular embryogenic structure on approximately 20% of the calli. Calli with such a compact
embryogenic structure were highly regenerable. When young inflorescences smaller than 0.75 cm were cultured, the embryogenic
structure yielded green plantlets with regeneration rates of 79.5% and 83.3%, respectively for the two cultivars. All 96 plants
regenerated from calli induced in the BA-containing medium were green and morphologically normal. The embryogenic nature of
the compact structure was confirmed by scanning electron microscopy.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
16.
Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO3 to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu2+ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA
ethylenediaminetetraacetic acid
- HPLC
high performance liquid chromatography
- MeOH
methanol
- MS medium
Murashige and Skoog medium
- WP medium
McCown's Woody Plant medium
- B5 medium
Gamborg B5 medium
- wt
weight 相似文献
17.
Heung Kyu Moon So Young Park Yong Wook Kim Sea Hyun Kim 《In vitro cellular & developmental biology. Plant》2008,44(2):119-127
An effective micropropagation technique via somatic embryogenesis has been developed using tissue from serially grafted shoots
generated from a mature Kalopanax septemlobus tree (~40 y old). Callus was induced from leaf segments obtained from the grafts by culturing the explants in Murashige and
Skoog (MS) medium supplemented with 2,4-D and 3% w/v sucrose under darkness. The effects of sucrose, coconut water, and polyethylene glycol (PEG-3350) were evaluated as factors
to promote development of somatic embryos (SEs) from embryogenic callus. More than 90% of explants formed callus; however,
only 2.5%, or 20 leaf segments out of 800 explants, formed embryogenic callus after 8 wk of culture. High sucrose concentrations
(3% and 5% w/v) were effective in inducing SEs. Treatment with 2–10% v/v coconut water also had a positive effect on embryo induction. A synergistic effect on SE induction was obtained using sucrose
and PEG, with presence of the latter compound resulting in smaller, more uniform SEs. Embryo germination and conversion to
plantlets were significantly influenced by the gelling agents. In general, gelrite-gelled medium was superior to agar-gelled
medium. In gelrite-gelled medium, gibberillic acid (GA3) enhanced embryo germination. Converted plantlets in an artificial soil mixture showed a 91% survival rate and displayed
no distinct morphological variations. Our results indicate that reliable somatic embryogenesis and plant production can be
achieved with rejuvenated tissues after repeated grafting of shoots derived from a mature Kalopanax septemlobus tree. 相似文献
18.
Dominique Laurain Jocelyne Trémouillaux-Guiller Jean-Claude Chénieux 《Plant cell reports》1993,12(9):501-505
Summary The present work establishes that isolated microspores of Ginkgo biloba L. cultured at densities of 1.5 to 5·104 per milliliter in Bourgin and Nitsch (1967) liquid medium are able to divide, both in the presence and in the absence of exogenous growth regulators, and to germinate by growing a pollen tube. In all experiments the microspores exhibited various modes of division leading to embryo formation in the liquid medium. Four weeks later, the microspores which had been previously submitted to various electrical stresses showed pro-embryo development earlier than those which had not. After ten weeks the number of embryos was found to be 300 to 5300 ml–1 following the experiments. When the embryos exhibited a slower growth in liquid medium, they were transferred onto various solid media for maturation. Two months later, embryos had proliferated visibly.Abbreviations BN
Bourgin and Nitsch (1967) medium
- IAA
Indole-3-acetic acid
- KIN
Kinetin
- GS
Growth substances 相似文献
19.
Experiments on three autumn-heading cauliflower genotypes (2 hybrids and a genotype selected from a population) were conducted to study different factors affecting anther culture. Culture conditions of the donor plants proved to be important: the best results were obtained during spring in a greenhouse where the temperature was maintained between 10 and 20°C. Overall winter and spring seemed more suitable than summer and early autumn for culture establishment. The optimal bud development stage depended on the genotype: for the hybrid 702, the greatest number of embryos for 100 plated anthers was obtained at the uninucleate pollen stage of the microspores; for V23.2 and 703, the optimal stage of the buds corresponded to the first mitotic division. Sucrose proved to be the best carbon supply for embryogenesis with an optimal concentration of 140 g l-1. The addition of a cytokinin (BAP) in the medium led to lower embryo production, and this negative effect increased when the hormone concentration in the medium increased. The use of liquid medium and a dark incubation period immediately after the high temperature treatment were favourable for embryogenesis. 相似文献
20.
Regulation of developmental pathways in cultured microspores of tobacco and snapdragon by medium pH 总被引:3,自引:0,他引:3
Barinova I Clément C Martiny L Baillieul F Soukupova H Heberle-Bors E Touraev A 《Planta》2004,219(1):141-146
The regulation of developmental pathways in cultured microspores of tobacco (Nicotiana tabacum L) and snapdragon (Antirrhinum majus L) by medium pH is described for the first time. Unicellular tobacco and snapdragon microspores developed into normal, fertile pollen when cultured in media T1 and AT3 at pH 7.0 and 25°C for 6 and 8 days, respectively. First, pollen mitosis was asymmetric and mature pollen grains were filled with starch granules and germinated upon transfer to a germination medium. However, when tobacco and snapdragon microspores were cultured in media T1 and AT3, respectively, at pH 8.0–8.5 for 4–6 days at 25 °C, the frequency of symmetric division increased significantly with the formation two nuclei of equal size, and the gametophytic pathway was blocked, as seen by the lack of starch accumulation and the inhibition of pollen germination. The transfer of these microspores to embryogenesis medium AT3 at pH 6.5 resulted in the formation of multicellular structures in both species and, in tobacco, in the formation of embryos and plants. In order to understand the possible mechanisms of the action of high pH, sucrose metabolism was analysed in isolated microspores of tobacco cultured at various pH values. Invertase (EC 3.2.1.26) activity in microspores was maximal at pH 5.0 and strongly decreased at higher pH, leading to a slow-down of sucrose cleavage. At the same time the incorporation of 14C-labelled sucrose from the medium into microspores was drastically reduced at high pH. These data suggest that isolated microspores are not able to metabolise carbohydrates at high pH and thus undergo starvation stress, which was shown earlier to block the gametophytic pathway and trigger sporophytic development. 相似文献