Identification of tetraploid regenerants from cotyledons of diploid watermelon cultured in vitro

Summary Adventitious shoots were obtained from the diploid watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] cultivars Dixielee, Jubilee II, Mickylee, Minilee, and Royal Sweet by culturing excised cotyledons on shoot regeneration medium for six weeks. Tetraploid and diploid regenerants were identified by counting the number of chloroplasts per guard cell pair from leaves of regenerated plants. Cross fertilization of putative tetraploids with diploid pollinators and the production of triploid seed confirmed the efficacy of this approach. The mean number of chloroplasts for tetraploid regenerants was 19.1 whereas diploids averaged 11.2. These values were similar to tetraploid and diploid plants from seed. Ovary diameter, petal, and anther diameter of male flowers, and leaf length by width ratio were also good indicators of plant ploidy. Progeny obtained from self-fertile tetraploids of Mickylee were crossed with various diploid pollinators to produce triploid hybrid seed. All triploid plants from tissue culture-derived tetraploids produced fruit comparable in quality to fruit produced by currently-available triploid hybrids, demonstrating that in vitro tetraploid induction can be used to produce high quality tetraploid plants for use in triploid hybrid seed production.     

Cytological studies on adventitious shoots and minitubers of a monoploid potato clone

Summary A three step procedure for adventitious shoot regeneration on leaf explants of monoploid potato clone H7322 and a minituber induction procedure on stem segments have been described. Chromosome counts on 92 adventitious shoots showed that 85% of them had been polyploidized, i.e., 71% were diploid, 1% tetraploid, and 13% were mixoploid. Cytophotometric studies on nuclei of soil grown tubers of tetraploid cv Astarte, of 1x, 2x and 4x adventitious shoots of H7322, and of diploid H²578 showed in all cases polyploidization with prominent classes up to 8C and 16C. However, nuclei of pith cells of 5 weeks old minitubers which had developed on monoploid H7322 itself or on 1x adventitious shoots of H7322 showed predominantly 1C and 2C values. Pith cells of minitubers of monoploid H7322 were screened, after iodine staining, for the presence of variant cells containing reddish-brown staining (amylose-free) starch. In more than 75% of the investigated minitubers one or a few of such variant cells were found indicating that such a variation occurs in minitubers of monoploid potato and that this variant character is expressed in cells of vegetative storage organs like potato tubers.     

In vitro Separation of Chimeral Pears into their Component Genotypes

Summary An adventitious shoot regeneration system for Pyrus communis was used to separate two chimeral pears into their component genotypes. The two cultivars were a variegated type, Louise Bonne Panachée and a red fruited mutant, Red Hardy. Leaves of these cultivars were placed onto a regeneration medium consisting of Nitsch & Nitsch (1969) salts supplied with various levels of Thidiazuron (TDZ) and NAA. After two months the regenerants were moved onto a proliferation medium of Lepoivre salts. Later they were evaluated for their chimeral status. Among the regenerants of the variegated type, 100% segregation occurred, most shoots were green, a few were albino. Regeneration was more efficient for dissociating the variegated chimera than rapid shoot multiplication and physical injury. In Red Hardy, after two months on the regeneration medium, 20 to 33% of the regenerants were green, the rest were red. The stability of the red and the green regenerants were assessed on media supplemented with various levels of sucrose and by total anthocyanins measurement. Both types were stable.     

Analysis of protoplast-derived plants of Saintpaulia ionantha H. Wendl

Of 3272 plants regenerated from protoplasts of 10 Saintpaulia ionantha genotypes, 98.4% survived transfer to the greenhouse. The frequency of regenerants with chlorophyll deficiencies, i.e. variegated leaves or albinos, was low (1.5%). There was a higher number of polyploid, in most cases tetraploid plants, regenerated from protoplasts (16%) which were identified by their altered morphology. Measurements of stomatal length and counting the number of chloroplasts per guard cell also allowed a clear differentiation between diploid and polyploid plants. The classification was confirmed by DNA content determination using flow cytometry. Mechanisms leading to polyploidization included spontaneous protoplast fusion as well as chromosome doubling during callus growth and shoot regeneration. Two genotypes with instabilities in flower colour showed completely altered flower colours in plants regenerated from protoplasts as well as in plants regenerated on leaf explants in vitro.     

二倍体、四倍体小麦成熟胚组织培养研究初报

以栽培二粒小麦(Triticum dicoccum Schuble)、硬粒小麦(Triticum durum Desf.)以及普通六倍体小麦祖先种野生二粒小麦(Triticum dicoccoides Kom.)、野生一粒小麦(Triticum aegilopides Bal.)4个基因型麦属植物成熟胚(MEs)为外植体,对成熟胚愈伤组织诱导、分化及植株再生的组织培养效果做了初步研究,筛选出适合于非六倍体小麦成熟胚组织培养的基因型.结果表明,不同基因型成熟胚愈伤组织的诱导、分化及植株再生差异显著,具有很强的基因型效应和基因型依赖性.其中栽培二粒小麦的成熟胚表现出较好的组织培养特性,它的愈伤组织诱导率、分化率、成苗率以及组培效率分别为95.00%、90.00%、32.40%、27.70%,显著高于其它基因型.二倍体、四倍体小麦成熟胚组织培养特性的研究以及组织培养基因型的筛选,为遗传转化体系的建立、小麦功能基因的分离和克隆,以及小麦分子育种工作奠定了基础.     

催眠睡茄的离体快繁及四倍体的诱导

以催眠睡茄腋芽为外植体建立快繁体系,利用秋水仙素进行催眠睡茄四倍体诱导,并将其与二倍体进行了初步的形态学和细胞学方面的比较.结果发现:催眠睡茄二倍体(2n=2x=48)最适增殖培养基为MS 6-BA 0.5 mg/L NAA 0.05 mg/L,生根培养基为1/2 MS IBA 0.2 mg/L;四倍体(2n=4x=96)诱导率因秋水仙素浓度和处理时间的不同而异,其中用0.5%的秋水仙素对丛生芽浸泡处理48 h诱导率最高,诱导率可达13.79%.四倍体与二倍体相比:叶片明显变厚,颜色加深,叶片气孔及其保卫细胞大小增大,气孔密度减小,且保卫细胞内叶绿体数目增加.     

A tetraploid hybrid plant from 4x × 2x crosses in Vitis and its origin

To study the origin of unreduced (2n) gametes in diploid Vitis cultivars, we surveyed the occurrence of tetraploid hybrid seedlings from 40 interploid crosses with five tetraploid and seven diploid cultivars. A total of 250 seedlings from the interploid crosses were established through embryo culture and by seed sowing. In 20 2x × 4x crosses, no tetraploid hybrid seedlings were derived from 8,902pollinations. In 20 4x × 2x crosses, two tetraploid hybrid seedlings were obtained from 8,057 pollinations. Investigation of isozyme genotypes of the two tetraploid seedlings using three variable enzyme systems indicated that one of the two seedlings resulted from the union of a diploid egg with a 2n male gamete and that failure of second meiotic division resulted in the formation of the 2n male gamete. The percentage of giant pollen grains in `Muscat Bailey A', a pollen parent of the tetraploid seedling, was relatively high(about 5.9%) and 10.9% of the giant pollen grains germinated on agar medium. These results suggested that 2n pollen of diploid cultivars are useful for breeding tetraploid grape. This revised version was published online in August 2006 with corrections to the Cover Date.     

4个优良石榴品种叶片高频再生体系的建立

为提高石榴叶片再生率,增加再生芽数量及缩短再生周期,以‘泰山红’‘、泰山三白甜’‘、皮亚曼’‘、牡丹’4个石榴品种盆栽苗和无菌苗叶片为试材,研究培养基类型、生长调节剂配比、叶片来源和培养步骤对不定芽再生的影响。结果表明：MS是叶片再生最适培养基类型;无菌苗叶片比盆栽苗叶片更易再生不定芽;BA3.0mg/L+KT1.0mg/L+IBA0.3mg/L是叶片愈伤组织诱导、分化和不定芽形成过程中生长调节剂最佳配比;先在液体培养基上培养3~5天,再转至固体培养基比仅在固体培养基上效果更好,叶片再生率最高达98.47%,出芽数达7.51个。叶片不定芽的最佳单芽培养基和增殖培养基分别为B5+BA0.6mg/L+IBA0.5mg/L+椰汁100mL/L+PVP2.0g/L+GA31.0mg/L和B5+BA0.8mg/L+IBA0.5mg/L+椰汁100mL/L+PVP2.0g/L,增殖系数为5.56。适宜的生根培养基为B5+IBA1.0mg/L+椰汁100mL/L,生根率为89.63%。由此建立了石榴叶片高频再生体系。     

墨兰多倍体的离体诱导和鉴定

以墨兰‘企剑白墨’的根状茎为材料,研究了秋水仙素处理对墨兰根状茎生长和多倍体诱导的影响。结果表明,秋水仙素对根状茎有严重的毒害致死作用,在试验浓度和时间范围内,随着秋水仙素浓度的升高或处理时间的延长,根状茎存活率明显降低。秋水仙素能诱导‘企剑白墨’根状茎的染色体加倍,但不同浓度和时间处理的染色体加倍效率不一样。只在0.01%×3 d处理下诱导出四倍体,诱导率为11.11%,其余处理诱导出二、四混倍体,诱导率在0～19.44%之间。四倍体根状茎比二倍体粗壮,顶端更圆,再生出的四倍体植株株型紧凑,叶片质地变硬,根明显增粗。     

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.     

不同基因型对花生胚小叶植株再生的影响

以不同类型18个花生品种成熟种子的胚小叶为外植体,对不定芽诱导及植株再生进行了研究,旨在为花生遗传转化和离体诱变等提供培养方法。将胚小叶外植体培养在添加1 mg/L NAA和6 mg/L BAP的诱导培养基上,4周后转移到添加4 mg/L BAP的培养基上进行培养。结果表明,所有的供试品种芽点诱导率均高于60%,但5大类型间及品种间(62.2%~95.5%)存在显著差异,龙生型平均诱导率最高(87.2%),其次是珍珠豆型(81.5%),多粒型最低(63.1%)。将形成芽点的外植体转移到添加4 mg/L BAP的培养基上后,部分外植体从芽点上分化出不定芽,继续培养不定芽伸长并再生植株。植株再生率也存在显著性差异,最高的是珍珠豆型(83.5%~97.1%),最低是多粒型(14.8%~22.0%)。     

Androgenetic plants of Anemone coronaria derived through anther culture

The genus Anemone (Ranunculaceae) includes many species cultivated for ornamental purposes. Most cut flower cultivars belong to A. coronaria L. and are multiplied by seed and sold for cultivation as 1‐year‐old tubers. As cultivars represent a population of hybrid individuals derived from crosses between heterozygous parents, the use of a true F₁ hybrid would improve the uniformity and quality of the product. As a first step towards the development of pure‐breeding lines, anther cultures were established from elite cultivars of A. coronaria. Somatic embryos and plantlets were regenerated from five elite cultivars, and up to 16.9 regenerants per 100 cultured anthers were obtained. Cytological analysis identified that 11 of 19 regenerants had either a 2x = 16 karyotype, or were mixoploids. RAPD‐based DNA fingerprinting showed that all the regenerants tested differed genetically from their anther donor, confirming their androgenetic origin. The shortening to 15 months for the time required to produce homozygous lines may convince seed companies to invest in F₁ hybrid breeding.     

非洲菊离体叶培养诱导不定芽研究

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

Histogenic analysis of chemically induced mixoploids in <Emphasis Type="Italic">Spathiphyllum wallisii</Emphasis>

Tetraploids and mixoploids were induced in several Spathiphyllum wallisii genotypes through in vitro application of mitosis inhibitors. Flow cytometry of leaves enabled the identification of sectorial hybrids, whereas microscopic nuclei research combined with root flow cytometry was required to provide insights in the histogenic composition of the mixoploids and to identify periclinal chimeras. Microscopic observation of epidermal or parenchymatic cell areas or the average cell thickness did not allow unequivocal characterization. However, direct visualization of diploid and tetraploid cells was enabled by analysis of nuclear areas. Root analysis was performed by classical fluorescence of metaphase chromosome spreads. This study showed the necessity of microscopic techniques for a thorough characterization of mixoploids. It also demonstrated the potential of the combination of these techniques with flow cytometry to unravel the exact effect of mitosis inhibitors on monocot plants. Finally, it might help to optimize the tetraploidization efficiency.     

Somatic hybrids between navel orange (Citrus sinensis) and grapefruit (C. paradisi) for seedless triploid breeding

Protoplasts from cell suspension cultures of ‘Bonnaza’ navel orange (Citrus sinensis (L.) Osbeck) were electrically fused with mesophyll protoplasts isolated from seedless ‘Red Blush’ grapefruit (Citrusparadisi). After 6 months of culture, a total of 20 plants were regenerated. Root tip chromosome counting revealed that 4 of them were tetraploids (2n = 4x = 36)and the rest were diploids (2n = 2x = 18) morphologically resembling the mesophyll parent. After 6 months of transplantation into the greenhouse, 4 of the diploidmesophyll regenerants unexpectedly flowered, but this phenomenon disappeared in the next year. This is the first report of precocious flowering in citrus via protoplast fusion. RAPD analysis further confirmed that the tetraploid regenerants were somatic hybrids while the diploid regenerants were mesophyll parent type. This somatic hybrid will be utilized as a possible pollen parent for improving the seedy pummelo cultivars in China by producing triploid seedless pummelo hybrid. The mechanism of early flowering was also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of genotype and light intensity on somatic embryogenesis and plant regeneration in melon (Cucumis melo L.)

The efficiency of 14 commercial cultivars of melon (Cucumis melo L.) for callus induction, plant regeneration and somatic embryogenesis under different photosynthetic photon flux densities (PPFDs) (150 or 50μmol m^?2 s^?1) was investigated. Cotyledonal explants were cultured on a Murashige and Skoog (MS) medium supplemented either with 9.0 μM 2,4-dichlorophenoxyacetic acid and 23.2μM kinetin or with 0.05 μM 2,4-dichlorophenoxyacetic acid and 0.26 μM 6-benzyladenine for the induction of somatic embryogenesis and shoots, respectively. For embryo maturation and root induction, growing callus tissues were transferred on growth regulator-free MS medium. Both genotype and the intensity of light significantly affected the rate of somatic embryo-genesis, embryo maturation and plant regeneration. On average, 12–47 primary globular-stage embryos were produced per mm² of explant surface. Fully developed, cotyledonary-stage somatic embryos were obtained from only three cultivars. Relatively high root induction rates were observed both on the shoot induction medium (11 cultivars) and on growth regulator-free medium (seven cultivars). In contrast, only six cultivars responded positively to the shoot induction treatment. Callus growth and somatic embryogenesis were significantly improved if cultures were incubated under higher PPFD values, although plant regeneration from all cultivars was significantly reduced under the same conditions.     

Induction of <Emphasis Type="Italic">Anthurium andraeanum</Emphasis> “Arizona” tetraploid by colchicine in vitro

Tetraploids plants of Anthurium andraeanum “Arizona” were successfully induced after treating diploid tissue masses with colchicine. Masses originating from diploid aerial roots were treated with colchicine at three different concentrations (i.e., 0.1, 0.2, 0.3%) for about 3, 5 and 7 h, and then were transferred into Murashige and Skoog medium containing 3 mg/l BAP + 0.2 mg/l 2,4-D. After 60 days, the survival rate and numbers of regenerative shoots were scored. The high concentration and longer duration sharply reduced survival rate. In contrast, the regeneration of plantlets was not noticeably affected by colchicine. Tetraploid plants were obtained in all treatments, but the percentage of induced tetraploids ranged from 0.2 to 7.6%. The best induction was obtained with a 5-h, treatment with 0.3% colchicine. The stomatal size of tetraploid plants was larger than in diploid plants; however, the stomatal density was lower than in diploid plants. Tetraploid plants possessed stronger petioles, thicker and deeper green leaves, and thicker and longer lived spathes in comparison with diploid plants. Abnormal spathes, such as double spathes or those lacking pedicels, were observed in tetraploid plants. Tetraploid plantlets could be regenerated via aerial roots; this technique could be applied to tetraploid plant propagation.     

Genomic constitutions and flower characteristics of selected Aranda orchid cultivars

Summary Ten Aranda cultivars commercially grown in Singapore were selected to study their genomic constitutions and flower characteristics. Cytological evidence and breeding records of these cultivars showed that they are of three genomic classes. Four of them are diploid with AV genomes (one Arachnis and one Vanda genome), another four are triploid with AVV genomes and the remaining two are tetraploid with AVVV genomes. Sizes of flowers as well as of sepals and petals generally show significant increases from diploid to tetraploid. This trend reflects the increasing influence of Vanda resulting from additional one and two Vanda genomes in triploid and tetraploid respectively as compared to diploid cultivars. Among the three genomic classes, diploid cultivars generally bear less flowers per spray than those of triploid and tetraploid although exceptions may occur. There is no clear trend in the length of inflorescences although diploid cultivars tend to have less compact spray with flowers more distantly spaced out.     

An assessment of the fertiliser response and other characters of three dihaploid potatoes and the characteristics of their tetraploid progenies

Summary Three dihaploids, two tetraploid cultivars and an unnamed tetraploid potato were subjected to three fertiliser treatments in a glasshouse. One dihaploid (PDH135), as well as being high yielding, responded to fertiliser dosage in a similar way to the cultivars by increasing tuber yield with each increase in fertiliser. The other two dihaploids increased shoot growth rather than tuber yield.An evaluation of tetraploid offspring obtained from dihaploid × tetraploid crosses, showed that PDH135 had the highest-yielding progenies in field trials. Other characters of the dihaploids were efficiently transferred into their tetraploid offspring. The use of dihaploids and unreduced gametes in breeding cultivars with useful quantitative characters is discussed.     

Barley lines showing prominent high green shoot regeneration in cultures derived from immature embryos

Eighty‐four cultivars of barley (Hordeum vulgare) and 95 wild strains (82 of H. spontaneum and 13 of H. agriocrithon) were surveyed for the production of callus, callus growth, and shoot regeneration in cultures derived from immature embryos. All cultivars except for ‘Turkey 381′, induced calli from more than 90% of embryos. On the other hand, the wild lines showed a large variation in the percentage of callus induction from 0 to 100%. Among the cultivars, those with the brittle rachis genotype, bt Bt₂, on chromosome 3H, regenerated shoots with a significantly higher percentage than the cultivars with the Bt bt₂ genotype. Green shoots were produced in a higher ratio (0.84) in the cultivars than in the wild lines (0.52). Among the lines examined,‘Lenins’ regenerated shoots efficiently (90.4%) and produced the highest number of calli with green shoots per embryo (4.77) followed by ‘Golden Promise’ (3.15). Examination of callus growth and shoot regeneration from embryos at different developmental stages revealed that scutellum development affected the quantity and quality of callus and shoot regeneration.