Highly efficient plant regeneration and <Emphasis Type="Italic">in vitro</Emphasis> polyploid induction using hypocotyl explants from diploid mulberry (<Emphasis Type="Italic">Morus multicaulis</Emphasis> Poir.)

Efficient plant regeneration is essential for successful transformation and in vitro polyploidy induction in mulberry. A high frequency (80%) of plant regeneration from hypocotyls occurred under in vitro conditions in mulberry (Morus multicaulis Poir.). We identified three key factors for enhancing successful regeneration based on earlier work: (1) hypocotyl position, (2) the combination and concentration of growth regulators, and (3) the addition of AgNO₃. The highest frequency of shoot regeneration was achieved using hypocotyl segments, which are proximal to apical meristems, and the optimal culture conditions were Murashige and Skoog’s (MS) (Murashige and Skoog, 1962) basal medium supplemented with 3.0 mg l⁻¹ 6-benzylamino purine, 0.3 mg l⁻¹ indole-3-acetic acid, 0.1% polyvinypyrrolidone, and 1.0 mg/l silver nitrate (AgNO₃) under subdued light at 25 ± 2°C. Treating the shoots with 0.2% colchicine (dipping for 72 h) resulted in a 14% tetraploid frequency, whereas a 20% tetraploid frequency resulted from using a 0.25% colchicine (dripping for 5 d) treatment, as determined by chromosome number counts. The diploid plant chromosome number was 28 (2n = 2x = 28) and that of tetraploid plants was 56 (2n = 4x = 56). Regenerated shoots rooted easily in 8–10 d using half-strength basal MS medium with 0.5 mg l⁻¹ indole-3-butyric acid and were successfully established in the soil.     

In vitro induction and identification of tetraploid plants of <Emphasis Type="Italic">Paulownia tomentosa</Emphasis>

Polyploidization is a major trend in plant evolution that has many advantages over diploid. In particular, the enlargement and lower fertility of polyploids are very attractive traits in forest tree breeding programs. We report here a system for the in vitro induction and identification of tetraploid plants of Paulownia tomentosa induced by colchicine treatment. Embryonic calluses derived from placentas were transferred to liquid Murashige and Skoog (MS) medium containing different concentrations of colchicine (0.01, 0.05, or 0.1%) and incubated for 24, 48, or 72 h on an orbital shaker at 110 rpm. The best result in terms of the production of tetraploid plantlets was obtained in the 48 h + 0.05% colchicine treatment, with more than 100 tetraploid plantlets being produced. The ploidy level of plantlets was verified by chromosome counts, flow cytometry, and morphology. The chromosome number of tetraploids was 2n = 4x = 80 and that of diploid plantlets was 2n = 2x = 40. The relative fluorescence intensity of tetraploids was twofold higher than that of diploids. The tetraploid and diploid plantlets differed significantly in leaf shape, with those of the former being round and those of the latter pentagonal. The mean length of the stomata was longer in tetraploid plants than diploid plants, and stomatal frequency was reduced with the increased ploidy level. The tetraploids had large floral organs that were easily distinguishable from those of diploid plants.     

In vitro induction of tetraploid plants from diploid Zizyphus jujuba Mill. cv. Zhanhua

Tetraploid plants of Zizyphus jujuba Mill. cv. Zhanhua were obtained with in vitro colchicine treatment. Shoot tips from in vitro-grown plants were treated with five different concentrations of colchicine (0.01, 0.03, 0.05, 0.1, 0.3%) in liquid MS medium (Murashige and Skoog 1962), and shaken (100 rpm) at 25 °C in darkness for 24, 48, 72 or 96 h, respectively. Tetraploids were obtained at a frequency of over 3% by using 0.05% colchicine (48 h, 72 h) and 0.1% colchicine (24 h, 48 h) treatment as determined by flow cytometry. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The chromosome number of diploid plants was 24 and that of tetraploid plants was 48. The stomata sizes of tetraploid plants were significantly larger than those of diploid plants, while the frequency of stomata were reduced significantly. Similarly, the chloroplast number of guard cells of tetraploid plants increased significantly. The selected tetraploid plants were grafted onto mature trees of Z. jujuba Mill. cv. Zhanhua in the field, resulted in thicker stems, rounder and succulent leaves, larger flowers and a delay in florescence time (3–4 days later) than diploid plants.     

In vitro tetraploid induction from leaf explants of Populus pseudo-simonii Kitag.

Tetraploid plants were produced from leaf explants of diploid Populus pseudo-simonii by treating the leaves with colchicine. Leaf explants were cultured on MS basal medium containing 1.78 μM BA and 1.08 μM NAA for 0, 6 and 12 days, and then transferred to the same MS liquid medium with colchicine at concentrations of 25, 50 and 75 μM for 1, 2 and 3 days. The highest efficiency of tetraploid induction was 14.6% by treating leaf explants that were pre-cultured for 6 days and then cultured in liquid MS with 50 μM colchicine for 3 days. Flow cytometric analysis was used to screen the tetraploids out from the regenerated plants and chromosome number counting was employed to confirm the polyploidy level. Size and frequency of leaf stomata between diploid and tetraploid plants were demonstrated to have significant differences.     

In vitro induction of polyploidy in Centella asiatica (L.) Urban

Mutational breeding was conducted using in vitro-grown shoot-tips of Centella asiatica (L.) Urban treated with colchicine (0.025–0.400% for 12–36 h) to induce polyploidy. Treated shoot-tips were grown on Murashige and Skoog (MS) medium supplemented with 4.54 μM thidiazuron (TDZ), and regenerated plantlets were acclimatized and transferred to soil. Two months following transfer to ex vitro conditions, ploidy levels of regenerated plants were determined by flow cytometry and by determining chromosome counts. Treating shoot-tips with colchicine concentrations ranging from 0.050–0.200% for 12–24 h promoted induction of tetraploids. Morphological and growth characteristics and the triterpenoid contents of the polyploids were also measured. Tetraploid plants demonstrated significantly longer stomata and a higher stomatal index compared to those of the diploid control plants. Furthermore, a positive trend in both biomass and triterpenoid production was obtained with the tetraploid and mixoploid plants of C. asiatica.     

Induction and identification of tetraploids using in vitro colchicine treatment of <Emphasis Type="Italic">Gerbera jamesonii</Emphasis> Bolus cv. Sciella

To induce variation through chromosome doubling in Gerbera jamesonii Bolus cv. Sciella, two-week-old in vitro grown shoots were treated with various concentrations of colchicine (0.01, 0.05, 0.10, 0.50 or 1% w/v) for 2, 4 or 8 h. Treated shoots were then cultured on Murashige and Skoog (MS) medium supplemented with 8.8 μM 6-benzyladenine (BA) and 155 μM adenine sulphate (ADS), and subsequently transferred to fresh MS medium containing 2.85 μM indole-3 acetic acid (IAA) for rooting. When shoots were treated with 0.1% colchicine for 8 h, 64% of recovered plantlets were tetraploid. Ploidy of plantlets was confirmed by flow cytometry, stomatal analysis, and morphological characters. Tetraploid plantlets displayed slower proliferation along with higher vigor and thickened broad leaves. Moreover, tetraploid plants developed larger flowers, longer stalks, and have improved vase-life, all contributing to higher ornamental value of gerbera.     

In vitro induction of tetraploids in Arachis paraguariensis

In this study, an efficient procedure was established for successful induction of tetraploid Arachis paraguariensis by treating diploid explants with colchicine. Quartered-seed, callus and shoot-tips were treated with colchicine at concentrations of 0.05, 0.1, 0.2 and 0.5?% (w/v) for 4, 8, 16, 20 and 24?h before they were transferred unto modified Murashige and Skoog medium for either callus induction or shoot regeneration. Results showed that quartered-seed displayed the highest frequency of in vitro plantlet regeneration and tetraploid induction, as well as the lowest mortality rate. Flow cytometric analysis also confirmed that the induced tetraploids from quartered-seed were true-to-type. The 0.5?% colchicine treatment for 4 to 8?h gave the best results with 39 and 43?% of the explants yielding tetraploid plants, respectively. Two?months following transfer to ex vitro environment, morphological and growth characteristics of the induced tetraploids were measured. Overall, increasing the ploidy level from 2× to 4× resulted in fewer stomata but more trichomes per unit leaf area. Tetraploid plants obtained in this study should expand the genetic base of Arachis, and can also be used in overcoming the existing hybridization barriers that may be due to ploidy differences within the genus Arachis.     

<Emphasis Type="Italic">In vitro</Emphasis> induction and identification of autotetraploids of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

Dioscorea zingiberensis is an important medicinal plant and a source of diosgenin in China. We report research on the induction, characteristics, and chemical assays of polyploid plants of D. zingiberensis. Immersing calli in 0.3% colchicine solution for 16 h prior to culture induced a high number of autotetraploid plants. The induction rate reached as high as 36.7% of treated calli. More than 50 lines of autotetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. Twenty selected tetraploid lines were transferred to the field for determination of morphological characteristics and for chemical assays. Six elite lines have been selected for further selection and breeding into new varieties for commercial production.     

In vitro induction, regeneration and analysis of autotetraploids derived from protoplasts and callus treated with colchicine in Citrus

In the present paper attempts were made to induce chromosome doubling of ‘Meiwa’ kumquat (Fortunella crassifolia) protoplasts and ‘Frost’ navel orange (Citrus sinensis Osbeck) embryogenic callus via colchicine treatment. Colchicine decreased protoplast viability, delayed protoplast division and inhibited callus growth, indicating presence of toxicity to cells. Cell lines established from ‘Meiwa’ protoplasts treated with 0.01 and 0.1% colchicine for 8, 16 and 24 h at each concentration showed different responses when they were cultured on embryoid-induction medium. Flow cytometry (FCM) demonstrated that tetraploids were detected in cell lines and embryoids from all of the treatments, with the highest frequency being 19.23%. As for ‘Frost’, tetraploid cells were only detected when the callus was treated with 0.1% colchicine for either 4 or 8 days, from which plantlets were regenerated. FCM and chromosome counting confirmed them as true tetraploids. The diploid cells were more active in mitotic division during a 12-day culture and smaller in size than their tetraploid counterpart. Potential applications of the novel tetraploid germplasms obtained through in vitro chromosome doubling to citrus cultivar improvement are discussed.     

野生南荻与芒杂种多倍体诱导研究

用不同浓度秋水仙素处理野生南荻×芒(Miscanthus lutarioriparia×Miscanthus sinensis)远缘杂交后代以诱导产生多倍体,并对变异株进行形态学和细胞学鉴定,以期获得稳定的四倍体植株并分析其生理特性。结果表明:(1)采用秋水仙素加入培养基处理法和秋水仙素溶液浸泡处理法都可获得一定频率的多倍体植株;胚性愈伤组织以0.2%秋水仙素浸泡处理48h的诱变效果较好,四倍体诱导率达8.7%;芽在0.05%秋水仙素培养基中处理15d较好,四倍体诱导率达10.6%;生根苗在0.1%秋水仙素培养基中处理10d较好,四倍体诱导率达11.1%。(2)经体细胞染色体计数,加倍植株染色体数为2n=4x=76,对照植株的染色体数目为2n=2x=38。(3)生长2年的多倍体植株形态、叶片大小、茎粗、茎壁厚、节间等性状表现出巨大性和超亲优势。     

In vitro induction and identification of autotetraploid of <Emphasis Type="Italic">Bletilla striata (Thunb.) Reichb.f</Emphasis>. by colchicine treatment

Polyploidy breeding has proved to be a valuable approach for acquiring the high yield superior varieties in medicinal plants. An effective protocol for obtaining Bletilla striata autotetraploid is in vitro induction of protocorms with colchicine. The protocorms of B.striata were soaked in different concentrations of colchicine solution [0.05, 0.1 and 0.2% (w/v)] for 12, 24, 36, 48 and 60 h, and the ploidy of the seedlings was identified by chromosome counting and flow cytometry analysis. The results showed that the optimal condition for induction of autotetraploid of B. striata protocorms was treated with 0.2% colchicine for 36 h with the induction rate reached as high as 26.7%. In addition, the morphological and anatomical characteristics were observed and compared between the diploid and tetraploid plants. And we found that the features of tetraploid plants were significantly different from diploid plants, such as tetraploid plants possessed thicker and deeper green leaves, larger stomata and more chloroplast number, which could be used as simple and efficient parameters for screening tetraploid. This study laid a foundation for breeding superior varieties of B. striata.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of protocorm-like bodies in <Emphasis Type="Italic">Cattleya</Emphasis>

A protocol for in vitro induction of crape myrtle tetraploids using nodes from in vitro-grown shoots (2n = 48) was established. Nodal buds were excised from in vitro-grown shoots, maintained on proliferation medium containing Murashige and Skoog medium supplemented with 4.44 μM 6-benzyladenine , 0.54 μM α-naphthaleneacetic acid, and treated with a range of concentrations of colchicine under three different conditions. Nodal bud explants treated in liquid proliferation medium supplemented with either 15 or 20 mM colchicine for 24 h turned necrotic and died; whereas, those cultured on solid proliferation medium supplemented with either 125 or 250 μM colchicine for 30 days survived, but no tetraploid plants were obtained. However, when explants were cultured in liquid proliferation medium containing 250, 500 or 750 μM colchicine for 10 days, tetraploid plants (2n = 96) were obtained. Incubation of explants in medium containing 750 μM colchicine promoted the highest frequency of survival (40%) of explants and of recovered tetraploids (60%). Morphological and anatomical characteristics of leaves, including leaf index, stomata size and number, stomata index (length/width), and number of chloroplasts in guard cells correlated with ploidy of crape myrtle plants. The number of chloroplasts in guard cells of stomata was a stable and reliable marker in discriminating plants of different ploidy levels. Chromosome counts and flow cytometry confirmed these findings.     

In vitro induction of tetraploidy in mulberry (Morus alba L.)

A high frequency of tetraploidy was induced in mulberry (Morus alba L.) through apical bud treatment under in vitro conditions. Apical buds from in vitro-grown plants were treated with three different concentrations (0.05, 0.1 and 0.2%) of colchicine in MS medium for 24 h. Tetraploidy at a frequency of 39.4±4.8% was obtained using 0.1% colchicine, whereas the frequency of tetraploidy was significantly reduced to 16.7±2.3% when 0.2% colchicine was used. Morphological, histological and cytological evidence indicated a phenotypic and genomic similarity of in vitro- with ex vitro-induced tetraploids. Rooting of tetraploids was on basal medium containing 2.6 μm NAA. The recovery of tetraploids was 80.8% more efficient using the in vitro method instead of the ex vitro method. The use of the same colchicine medium for up to 4 weeks with additional explants was found to be equally effective for the induction of tetraploidy. Received: 6 January 1997 / Revision received: 6 October 1997 / Accepted: 12 December 1997     

Induction of tetraploidy in Buddleia globosa

The aim was to produce a tetraploid form of Buddleia globosa to facilitate introgression of yellow flower colour into B. davidii, which is naturally tetraploid. Protocols were established for the micropropagation of B. globosa and tetraploid plants were obtained by application in vitro of colchicine to pre-cultured excised nodal sections. Three concentrations of colchicine were applied (0.01%, 0.05% and 0.1% w/v) for 1, 2 or 3 days. At 0.01% tetraploids were produced only after 2 days of application. All other treatments produced at least one tetraploid. The colchicine technique was extremely effective: of 29 lines tested, 19 were tetraploid and 5 were mixoploid. The vegetative characteristics of these tetraploids are described and the flowering characteristics of the three that flowered. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro production and identification of autotetraploids of Scutellaria baicalensis

The roots of Scutellaria baicalensis are a major traditional Chinese medicine. We report research on induction, characteristics and chemical analysis of polyploid plants of S. baicalensis. Immersing calluses in 0.2% colchicine solution for 12 h prior to culture induced a high number of tetraploid plants. The induction rate reached as high as 40% of treated calluses. More than 50 lines of tetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. Twenty selected tetraploid lines were transferred to the field for determination of morphological characteristics and for chemical assays. Seven elite lines have been selected for further selection and breeding into new varieties for commercial production.     

Comparative and evolutionary analysis in natural diploid and tetraploid weather loach Misgurnus anguillicaudatus based on cytochrome b sequence data in central China

To obtain the phylogenetic relationship between diploid and tetraploid Misgurnus anguillicaudatus, the mitochondrial cyt b gene in the diploid and tetraploid weather loach were isolated and sequenced. The DNA sequences were analyzed using MEGA 3.0 software to determine the phylogenetic relationship. Forty-five variable sites among cyt b gene sequences and 18 amino acid substitutions occurred within the diploid and tetraploid loaches as deduced from the nucleotide sequences analysis of the cyt b gene. The nucleotide pairwise distance between diploid and tetraploid loach ranged from 0.001 to 0.025. Phylogenetic analysis revealed evolutionary relationships between diploid and tetraploid loach. Our results indicated a significant difference between diploid and tetraploid loach about the cyt b gene. AMOVA analysis indicated that there were no significant genetic variations within diploid loaches (Fst = 0.2529, P > 0.05) and within tetraploid loaches (Fst = 0.0564, P > 0.05), neither. However, significant genetic differences were found between diploid and tetraploid loaches (Fst = 0.7634, P < 0.05). Thus, it is concluded that no reproductive isolation was found within the same cytotypes of different localities, but there was reproductive isolation between these two cytotypes. The diploid loach existed before the tetraploid loach in nature. The present study is the first to describe the phylogenetic relationships of natural polyploidy weather loach using mtDNA cyt b gene.     

Enhancement of paclitaxel content in induced tetraploid <Emphasis Type="Italic">Corylus avellana</Emphasis> L. cell suspension culture with regulating the expression of genes in paclitaxel biosynthetic pathway

During recent years, hazel cell suspension culture has been significantly considered as a new important source of paclitaxel. Artificial polyploidy alters different characters in plants which results in amplifying the secondary metabolites in medicinal plants. In this paper, the effects of tetraploidy induction on paclitaxel content and gene expression in hazel cell suspension culture were investigated. Various concentrations of colchicine and duration of exposure in solid and liquid media were examined and the ploidy level of cells was determined using flow cytometric analysis. The tetraploid cells were obtained from 0.2% colchicine in the exposure time of 5 and 6 days in solid medium and 0.3% colchicine in the exposure time of 3 and 4 days in liquid medium. Tetraploid cells were employed to prepare cell suspension. 3 µM of phenylalanine and 0.05 mM of vanadyl sulfate were added to both tetraploid and diploid (control) suspension to elicit paclitaxel induction. High performance liquid chromatography analysis demonstrated that the tetraploid cell suspensions produced paclitaxel of about [9.88 µg g^?1 (DW)] 1.7-fold compared with diploid cells [5.74 µg g^?1 (DW)]. The application of phenylalanine and vanadyl sulfate increased the concentration of paclitaxel in both diploid and tetraploid cells. Moreover, qRT-PCR analysis showed that the expression of GGPPS gene was significantly increased and PAL gene expression was altered after tetraploidization.     

In vitro induction of polyploidy in yacon (<Emphasis Type="Italic">Smallanthus sonchifolius</Emphasis>)

In vitro chromosome doubling was induced in octoploid (2n = 58) yacon using oryzalin and colchicine as mitotic spindle inhibitors. Nodal segments of in vitro cultured plants, 5–15 mm long, were exposed to 20, 25, or 30 μM oryzalin and 1, 3, or 5 mM colchicine for 24 or 48 h. The resulting ploidy level was determined by chromosome counting and flow cytometry. Out of 240 nodal segments, 3.33% hexadecaploid (2n = 116) plants were regenerated after the application of oryzalin. The greatest proportions of hexadecaploid plants (1.6%) were obtained after 48 h of 25 μM oryzalin treatment. With the colchicine treatment, only 0.42% hexadecaploid plants were detected and their survival rate was significantly lower in comparison with the oryzalin treatment. In hexadecaploid yacon, significantly higher levels of saccharides were detected (FOS 13.9 g/100 g FM, fructose 4.6 g/100 g FM and glucose 2.1 g/100 g FM) compared to the octoploid control (FOS 5.3 g/100 g FM, fructose 2.9 g/100 g FM and glucose 1.0 g/100 g FM). These results indicate that in vitro treatment of nodal segments with oryzalin solution could be an effective procedure for chromosome doubling and the polyploidy breeding can help to increase the FOS content in the tuberous roots.     

Duplication of the chromosome number of diploid Brachiaria brizantha plants using colchicine

 Some species of Brachiaria, generally tetraploid apomictic varieties, have become important forage grasses in the tropics. Breeding of Brachiaria depends on compatibility with the available apomitic tretraploid cultivars. This paper describes a procedure for chromosome duplication of two Bracharia brizantha diploid sexual accessions, using colchicine treatment of basal segments of in-vitro-grown plants. Explants were cultured on a medium containing 1 mg/l naphthaleneacetic acid, 3 mg/l kinetin and 0.01% colchicine for 48 h and transferred to the same medium without colchicine until shoot regeneration occurred. Regenerated plants were screened by flow cytometry, and chromosome number duplication was confirmed by cytological analysis of root tips. Received: 9 Ocotber 1997 / Revision received: 9 December 1998 / Accepted: 15 June 1999     

Production of tetraploid plants of non apomictic citrus genotypes

Ploidy manipulation in Citrus is a major issue of current breeding programs aiming to develop triploid seedless mandarins to address consumer demands for seedless fruits. The most effective method to obtain triploid hybrids is to pollinate tetraploid non apomictic cultivars with pollen of diploid varieties. Such non apomictic tetraploid lines are not found in the citrus germplasm and need to be created. In this work we describe a new methodology based on in vitro shoot-tip grafting combined with treatment of the micro-grafted shoot-tip with colchicine and oryzalin to achieve chromosome doubling and a dechimerization procedure assisted by flow cytometry. Stable tetraploid plants of Clemenules, Fina and Marisol clementines and Moncada mandarin have been obtained directly from shoot tip grafting combined with colchicine and oryzalin treatments or after dechimerization of mixoploids plants (2x–4x). These stable tetraploid plants have been used in 4x × 2x hybridizations, to recover over 3,250 triploid hybrids in 3 years.