In vitro chromosome doubling of Miscanthus sinensis

The aim was to develop an efficient chromosome doubling method for Miscanthus sinensis to enable the production of triploids and so avoid seed dispersal to the environment. Antimitotic treatments with colchicine or oryzalin were tested in M. sinensis cl. MS‐88‐110 on: (1) in vitro shoots and plants established in soil; (2) during propagation of embryogenic callus; and (c) during the initial stages of callus induction. All systems produced chromosome‐doubled plants. A higher percentage of tetraploids was found after antimitotic treatment at the explant or callus level compared with treatment of in vitro shoots or plants established in soil. In general, oryzalin was more toxic to plant material than colchicine. A higher frequency of chimeras was found among plants with altered ploidy level when the target was formed shoot buds compared with adventitious shoot formation from callus. Antimitotic treatment of embryogenic callus from shoot apices also resulted in a high degree of albinism.     

Chromosome doubling of androgenic haploid plantlets of rice (Oryza sativa) using antimitotic compounds

The regeneration of haploid plantlets is considered as a bottleneck in rice anther culture. In this study, an antimitotic chromosome doubling method, simple and efficient, of androgenic haploid plantlets resulted in an efficient doubled haploid obtainment. Through chromosome doubling capacity comparison of the three antimitotic compounds (colchicine, trifluralin and oryzalin), colchicine at 500 and 625 mg/L without supplementing with DMSO was found to be the best antimitotic treatment, with a chromosome doubling capacity of 40%. Furthermore, the in vitro growth of plantlets was followed to analyse the effects of antimitotic compounds. Colchicine treatments were more toxic than dinitroanilines, and colchicine DMSO-supplemented treatments had significant lower values on shoot growth. On the other hand, dinitroaniline compounds impeded root growth, provoked helical growth of shoot and caused the apparition of white nodules in the base of the plantlet due to sprouting abortion. In this study, a protocol for doubled haploid plant recovery was established taking advantage from androgenic haploid plantlets in order to increase the number of doubled haploid plantlets produced after an anther culture protocol.     

Oryzalin as an Efficient Agent for Chromosome Doubling of Haploid Apple Shoots in vitro

In an outbreeding species such as apple, haploid plants may be especially useful in breeding programmes for the production of homozygous material. However, methods must be available to induce chromosome doubling in the haploid plants. Two antimitotic agents, colchicine and oryzalin, were compared as regards their efficiency in inducing chromosome doubling of in vitro haploid apple shoots. Three colchicine levels (0.025, 0.25 and 1.25 mM) and three oryzalin levels (5, 15 and 30 μM) were evaluated. Three techniques were also used and compared. Survival rate and chromosome counts were determined. Differences were observed between the two antimitotic agents and between the three techniques. This study demonstrates that oryzalin could be a better choice than colchicine for chromosome doubling on haploid apple shoots in vitro.     

Antimicrotubule herbicides for in vitro chromosome doubling in Beta vulgaris L. ovule culture

In vitro chromosome doubling during ovule culture of sugar and fodder beets (Beta vulgaris L.) was studied with four anti-microtubule herbicides: amiprophos-methyl (APM), oryzalin, pronamide, and trifluralin at concentrations of 0–300 μM. Best chromosome doubling results were obtained by treatment of the ovules with 100 μM APM which produced 4.7 diploid plants per 100 ovules. Highest chromosome doubling was found with oryzalin using 1 μM, with trifluralin at 10 μM, and with pronamide at 10 μM producing 2.8, 2.0, and 2.0 diploid plants per 100 ovules, respectively. The APM treatments showed relatively low toxicity on embryo formation which in combination with a high chromosome doubling effect, resulted in up to 89 diploids per 100 plants regenerated. Oryzalin and trifluralin had more severe toxic effects, which reduced embryo formation, thereby lower percentages of chromosome doubled plants were obtained from these treatments. Pronamide had no significant toxic effect but it induced chromosome doubling at lower frequencies. Compared to colchicine, APM seems to be as efficient for chromosome doubling during beet ovule culture, but at molar concentrations 100 times lower than those used for chromosome doubling with colchicine. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficient Production of Doubled Haploid Plants through Chromosome Doubling of Isolated Microspores in Brassica napus

Three methods of chromosome doubling to produce doubled haploid plants from microspore cultures of Brassica napus were compared: colchicine treatment of microspore-derived plants, microspore-derived embryos, and isolated microspores. In the whole plant treatment, 53% of the treated plants set seed, but the treatment delayed plant growth and reduced seed set. When microspore-derived embryos were treated with colchicine, the doubling frequency was 32% (compared to 15% for spontaneous doubling). Direct colchicine treatment of isolated microspores resulted in a doubling efficiency of 70 % of the whole plants. This treatment also stimulated embryogenesis in microspore culture, leading to increased plant regeneration. Thus, direct chromosome doubling of isolated microspores is efficient and more than 10 000 doubled haploid plants have been produced in this manner in the past three years in order to accelerate the plant-breeding process.     

Polyploidization and early screening of Rhododendron hybrids

Polyploid induction represents a useful tool for breeders of floral crops as larger flowers, longer flowering period and deeper colors can be achieved through chromosome doubling. This study aimed at testing the efficiency of colchicine and oryzalin in inducing polyploidy in three Rhododendroncultivars grown in vitro. The chemicals were used in two concentrations with 24 h and 48 h treatment durations. The survival of the plants was better in colchicine than in oryzalin solutions. The higher concentration of both chemical skilled more plantlets. The treatment duration in oryzalin did not affect the survival, but 48 h in colchicine was more destructive than 24 h. The low survival rate may not be a disadvantage, if the treatment induces desired ploidy. The ploidy levels were screened with flow cytometry. Oryzalin was more efficient than cochicine in inducing polyploidy, the treatment duration and the concentration did not have significant effects as main factors. The biggest proportion of solid tetraploids (18.2% of the survived plants) was obtained from the 24 h treatment in 0.005% oryzalin. Immediately after the treatment the polyploids grew very slowly, whereas most of the unaffected diploids were vigorous from the very beginning. More mixoploids than solid tetraploids were obtained in all treatments. Most of the mixoploids retained their chimerism, one third shifted todiploidy and one single plant to tetraploidy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spontaneous and induced chromosome doubling in gynogenic lines of onion (Allium cepa L.)

The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) and 6-benzyladenine (6—BA) in the medium is required to induce an acceptable yield of gynogenic embryos from unfertilized ovary and flower cultures in onion. Four different exposure times of ovary and flower cultures to exogenous growth regulators (15, 30, and 45 days, and the entire culture period) were assayed. The objective was to ascertain the effect of these substances and of their period of application on the formation of gynogenic embryos and on the yield of haploids. An exposure of 15 days was sufficient for ovaries and flowers to be stimulated towards the gynogenic response, whereas, for the remaining period of 30—80 days, the pro-embryos could easily grow on a growth-regulator-free medium. In the gynogenic material obtained, phenotypic differences were visible between genetically independent lines but not between plants within each line, even when they had a different ploidy level (n or 2n). Almost all lines obtained by gynogenesis were sterile. Only a small percentage (1%) became fertile through spontaneous chromosome doubling, and these produced 2—20 seeds each, giving normal plants. The recovery of fertility occurred more often during the generations of bulbils. To exploit this natural propensity towards diploidization in this phase, different amounts and numbers of applications of colchicine were evaluated in two experiments. The treatments corresponding to 10 and 100 mg/1 of colchicine applied for 24h gave the highest number of diploid cells in root tips but no diploidization occurred in the shoot apices. Three days of colchicine treatment at 10 mg/1 produced 46% of plants completely diploid in the shoot apex. The flower fertility of these doubled haploid plants is being evaluated.     

Development of novel chromosome doubling strategies for wheat × maize system of wheat haploid production

Two chromosome doubling strategies were evaluated for producing wheat doubled haploids from wheat x maize crosses: (i) in vitro colchicine application to haploid embryos and (ii) colchicine treatment through postpollination tiller injections. In the in vitro approach the haploid embryos were rescued on medium containing colchicine (at concentrations of 0.2, 0.3, 0.4 and 0.5%) and moved to a colchicine‐free regeneration medium 48 h later. Embryos exposed to 0.5% colchicine had 91.67% of their regenerated plants showing chromosome doubling. In the tiller injection approach, different concentrations (0.5, 0.75 and 1.0%) of colchicine solution, which also contained 2,4‐D (100 ppm), were injected into the uppermost inter‐node of crossed tillers 48 and 72 h after pollination. The chromosome doubling efficiency varied from 33 to 100%, with 1% treatment being the most effective. No chimeras of doubled/haploid sectors were observed in the case of the tiller injection treatment and all the florets showed seed set in the doubled plants. Stomatal guard cell length provided rapid, early‐stage and unambiguous analysis of ploidy level on the basis of 10 guard cell observations per plant.     

Artificial polyploidy in medicinal plants: Advancement in the last two decades and impending prospects

Medicinal plants are in huge demand since the consumption is widespread and ever-increasing globally. The conventional breeding programs are generally environmental dependent; prone to different biotic and abiotic stresses as well as the secondary metabolite content is too low to harvest. In this context, developing polyploid individuals artificially would be a remarkable approach to increase vigor and attain this objective. Polyploids often exhibit some morphological features that are different or greater in forms than their diploid progenies. Polyploidization can be induced by quite a few antimitotic agents. The most frequently used antimitotic chemicals are colchicine, trifluralin, and oryzalin. The whole method of induced chromosome doubling consists of a series of steps, including an induction phase, regrowth phase, and a confirmation technique to evaluate the rate of achievement. The induction phase depends on different factors, such as explant types, antimitotic agents, its different concentrations, and exposure durations. To evaluate the accomplishment of polyploidization, morphological or anatomical observations are recorded as a rapid method. However, chromosome count and flow cytometry are the most eminent method for absolute confirmation. Despite significant prospects of polyploidization, there has been very little research on medicinal plants. The current review gives an overview of the different parameters of in vitro chromosome doubling, the history of the technique, and progress made over the last two decades.     

Intra‐ and inter‐specific crosses of Solanum materials after mitotic polyploidization in vitro

Mitotic polyploidization in vitro was used in selected wild Solanum species and Solanum tuberosum dihaploids. The efficiency of polyploidization by colchicine was compared with that of oryzalin. Oryzalin was more effective than colchicine (P = 0.1). The rate of non‐affected to mixoploid to tetraploid regenerants was 22 : 2.5 : 1 (colchicine) and 14 : 2 : 1 (oryzalin). Optimal concentrations and durations were 3.5 mm /24 h for colchicine and 25 or 30 μm for 24 or 48 h for oryzalin (variations in concentration and duration are necessary owing to possible diversity of responses in selected genotypes). Tetraploids were obtained from S. berthaultii, S. bulbocastanum, S. pinnatisectum, S. verrucosum and eleven S. tuberosum dihaploids. The yield of tetraploids derived from tbr dihaploids was lower than that from the wild species (P = 0.01). Tetraploid regenerants were tested in intra‐ and inter‐specific crosses. Three of 43 intra‐specific combinations (298 pollinated flowers) were successful and yielded 440 seeds. Inter‐specific crosses (138 combinations, 1672 pollinated flowers) yielded 48 seedless berries.     

Doubling the chromosome number of bahiagrass via tissue culture

Crop improvement in bahiagrass (Paspalum notatum Flüggé) is limited by apomixis in most natural tetraploids, however, diploid sexual types occur. Production of sexual tetraploids by chromosome doubling will allow hybridization with apomictic tetraploids. Diploid bahiagrass (Paspalum notatum Flüggé) embryogenic callus tissue was exposed to three concentrations of three antimitotic chemical agents, colchicine, oryzalin and trifluralin. Callus was generated to plants and ploidy was evaluated by stomata size, mitotic chromosome counts, and flow cytometry. A total of 310 plants were verified as tetraploid of 1,432 plants that reached transplanting size. All treatments yielded 4x plants. The mean percentage success over all treatments was 22%, with means of 31% for oryzalin, 24% for colchicine and 16% for trifluralin. The high rates of success indicate that all agents can be successfully used to double chromosome numbers in bahiagrass. The percentage of 4x plants ranged from 9% (20 μM trifluralin) to 43% (20 μM oryzalin). Several treatments adversely affected regeneration. Mitotic chromosome counts are difficult and labor intensive in bahiagrass. Therefore, leaf stomata measurements were used as a preliminary screen. Data gave a bimodal distribution with overlapping tails and based on chromosome counts would have given an error rate of 12%. Flow cytometry analysis of regenerated plants resulted in mean nucleus fluorescence distributions consistent with control diploid or tetraploid values. These values agreed with chromosome counts, and this method is recommended for determining bahiagrass ploidy level. Research goals and available resources should be taken into consideration when selecting a treatment for chromosome doubling in bahiagrass.     

Induction and verification of autotetraploids in diploid banana (Musa acuminata) by in vitro techniques

Summary Flow cytometry and stomata characteristics were used for screening ploidy levels in a large population of in vitro induced autopolyploids of the Musa acuminata breeding clone SH-3362. Culturing shoot tips in liquid medium stipplemented both with 5.0 mM colchicine for 48 hours or 30 M oryzalin (3,5-dinitro-N4,N-dipropylsulphate) for seven days, both in combination with 2% (v/v) DMSO, resulted in a high (23.1% and 29.1%) frequency of non-chimeric tetraploids in the fourth vegetative generation. Although mixoploidy persisted in subsequent cycles of vegetative propagation, tetraploids as identified by flow cytometry remained solid non-chimeric during two more cycles. These autotetraploids were propagated for field testing. A rough pre-selection of regenerated V4 plants based on their stomata characteristics resulted in a population in which only 56.2% of the plants were solid tetraploids. The somatic polyploidization system reported here can be utilised for banana breeding programmes.Abbreviations FCM Flow Cytometry     

芦笋单倍体染色体加倍技术研究

研究了芦笋单倍体染色体加倍的方法。在离体培养条件下,以秋水仙素为诱变剂,分别用浸泡法和培养基添加法处理芦笋单倍体幼苗的茎尖,比较了秋水仙素不同浓度、不同处理时间的诱导效果。结果表明,培养基添加法的诱导效果好于浸泡法,当在培养基中添加0.3%秋水仙素并处理7天时诱导效果最佳,染色体加倍频率与成活率分别可达82.50%和80%。加倍后的二倍体植株与单倍体植株相比,茎干变粗,气孔与保卫细胞增大,保卫细胞内的叶绿体数增多。     

Chromosome doubling of mature haploid Brussels sprout plants by colchicine treatment

Summary The effect of colchicine concentration, and the mode and time of colchicine application in doubling the chromosomes of haploid Brussels sprout plants was assessed by scoring treated plants for the presence of diploid flowers and seed set after self-pollination.When colchicine treatments were applied after the plants had been vernalised, using 2 dose rates and 3 methods of application, only 38.1% became doubled and only 13.8% produced seed. Treatment with 0.1% colchicine with or without the addition of 2% dimethyl sulphoxide gave doubling rates similar to those reached with 0.05% colchicine, but resulted in more damage to the apices.When 0.05% colchicine solution was injected into the plants' apices at varying times during vernalisation, the rate of doubling was 71.2% on average and 50.7% of plants gave seed on selfing.Overall doubling rates of plants where the apex was easily accessible were 79.0%, while for plants with dense terminal heads they were 35.0% and for plants with visible buds 31.4%.     

The effect of colchicine on triticale anther-derived plants: Microspore pre-treatment and haploid-plant treatment using a hydroponic recovery system

In cereals, chromosome doubling of microspore-derived haploid plants is a critical step in producing doubled haploid plants. This investigation was undertaken to study the effect of incorporation of colchicine in the induction medium for anther culture, and the effect of colchicine on anther culture-derived plants of triticale grown under controlled greenhouse conditions. In the latter case, chromosome doubling of adult sterile plants derived from anther culture of fourteen triticale populations was attempted, where androgenetic plants with non-dehiscent anthers were cloned and subjected to the colchicine treatment, and then grown with the aid of hydroponics. The hydroponic system provided optimal conditions for recovery of the affected haploids from the toxic effects of colchicine treatment and all colchicine-treated plants survived. A topcross-F₁ (TC₁F₁) population with timopheevii cytoplasm produced the highest percentage of plants with seed-set either due to chromosome doubling by colchicine (98%) or spontaneous doubling of chromosome number (15%). Colchicine-treated anthers performed inferior than control in both induction and regeneration phases. One of the key observation of this study was the reversal from reproductive stage back to the vegetative stage which in turn enabled further cloning of haploid plants under hydroponic conditions once they were identified as sterile. The one hundred percent survival rate of in vitro-derived plants, 100% survival rate of colchicine treated haploid plants and the high chromosome doubling success rate (X = 82.3) observed in this study imply that a temperature-controlled greenhouse with an hydroponic system provides an efficient environment for inducing chromosome doubling of haploid plants in cereals. This revised version was published online in August 2006 with corrections to the Cover Date.     

Trueness-To-Type and Yield Components of the Banana Hybrid Cultivar FHIA-18 Plants Regenerated Via Somatic Embryogenesis in a Bioreactor

Summary A population of 1,500 plants of the banana hybrid ‘FHIA-18’ (AAAB), regenerated from somatic embryos, which were multiplied in bioreactors, showed similar characteristics to plants propagated from shoot tip cultures both in the acclimatization stage and in field experiments carried out in Cuba. The plants originating from somatic embryos were similar to the plants obtained from shoot tips with respect to plant height, diameter of the pseudostem and number of suckers. Both groups of plants obtained from in vitro cultures were significantly different to the plants obtained from suckers during the flowering period of the mother plants, which was shortened by two months. The greater plant height and diameter of the pseudostem in the plants coming from somatic embryos and shoot tip is due to the effect of in vitro culture, and this was observed in different banana and plantain cultivars. During the second cycle of evaluation, the plants coming from the three propagation methods studied in this work had similar growth habits without significant differences in the majority of the morphological parameters evaluated. These results confirm that the difference obtained during the first cycle between the distinct populations is attributed to temporary changes. The original characteristics of the cultivar were evident from the second cycle of culture. Only 0.13% somaclonal variant was observed in the plants coming from somatic embryogenesis. These percentages are low taking into consideration that other propagated methods accept up to 5% variants in field conditions.     

In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine (Vitis vinifera L.)

A protocol for in vitro induction of tetraploids via colchicine-treated somatic embryos from immature zygotic embryos of diploid grapevine (Vitis vinifera L.) is reported. Embryogenic callus was initiated from immature zygotic embryos cultured on Nitsch and Nitsch (NN) medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The callus was transferred to NN medium containing 1.0 mg/l α-naphthalene acetic acid (NAA) and 0.5 mg/l benzyladenine (BA) to establish somatic embryogenesis. The vigorously growing globular embryos were selected and treated by 0, 10 or 20 mg/l colchicine for 1, 2 or 3 days, and then immediately transferred to NN medium supplemented with 0.03 mg/l NAA and 0.5 mg/l BA, for somatic embryo conversion and plant regeneration. The number of surviving embryos and regenerated plantlets following colchicine treatment decreased with increasing colchicine concentration and treatment time. Among 29 randomly investigated plantlets regenerated from colchicine-treated somatic embryos, five solid tetraploids (2n = 4× = 76) were identified by chromosome counting analysis; all others were diploid (2n = 2× = 38). Ploidy level of plant regenerated was also determined from leaves using flow cytometry. No chimeras with both 2C and 4C nuclei was produced from colchicine-treated somatic embryos. Significant differences in leaf stomata parameters were observed between diploid and induced tetraploid plantlets.     

Efficient in vitro Chromosome Doubling During Beta vulgaris Ovule Culture

The effect of in vitro colchicine treatment of sugar beet ovules, after 7 days culture, on embryo formation, regeneration and ploidy of regenerated plants was studied with 5 concentrations of colchicine and 5 durations of treatment arranged as a 5 × 5 factorial in incomplete blocks. The best results were obtained with the shortest duration of treatment (5 hours) and the highest concentration of colchicine (0.4 %) giving 5.0 diploid plants per 100 ovules with 62.1 % of regenerated plants being diploid. Statistical analysis revealed that treatment effects could be separated into a toxic effect reducing embryo formation and a chromosome doubling effect affecting percentages of diploid regenerated plants. Toxic effects on embryo formation could be explained by simple exponential decay models, toxicity of the drug (decay constant) increasing linearly with duration of treatment. Duration of treatment had no effect on chromosome doubling percentages. The effects of colchicine concentration on chromosome doubling were explained by an exponential saturation model with spontaneous chromosome doubling of 8.1 % and saturation at 51.4 % diploid plants at 0.2 % and higher colchicine concentrations. In addition, treatments increased percentages of 4N and 6N plants from 0 % without colchicine to 10 % on average for treated ovules. A response surface model fitted to the total yield of diploid plants per ovule indicated that shorter durations of treatment and higher colchicine concentration may improve results.     

The production of homozygous diploid plants of Solanum verrucosum by tissue culture techniques

Summary Monohaploid plants of S. verrucosum (2n=x=12) were induced in anther culture. Axillary buds from these plants were treated with colchicine in shoot tip culture for 48 hours and then transferred to a colchicine free medium. The resulting plantlets were scored for diploidy by stomatal chloroplast counts and root tip cytology and it was found that doubling of the chromosome number had occurred.     

In vitro induction of tetraploid plants from callus cultures of diploid bananas (<Emphasis Type="Italic">Musa acuminata</Emphasis>, AA group) ‘Kluai Leb Mu Nang’ and ‘Kluai Sa’

Tetraploid plants were induced successfully from diploid bananas Musa acuminata (AA genome) ‘Kluai Leb Mu Nang’ and ‘Kluai Sa’ (2n = 2x = 22) with in vitro oryzalin treatment. Calluses from in vitro-grown shoot tips of both cultivars were treated with oryzalin at concentrations of 1.5 or 3 mg l⁻¹ for 24, 48 and 72 h, respectively. The oryzalin treatments produced tetraploids at a frequency of 15.6% in ‘Kluai Leb Mu Nang’ and 16.7% in ‘Kluai Sa’ as detected by flow cytometry. Chromosome counting showed that the tetraploid plant chromosome number was (2n = 4x = 44). The selected tetraploid plants were transplanted in the field and variations in the morphological characteristic of leaf shape and fruit bunch compared to normal diploid plants were found under the same growing condition even after 3 years of cultivation.