Production of triploid plants of mulberry (Morus alba L) by endosperm culture

 A continuously growing callus was obtained from immature endosperm of Morus alba L Cv S-36 cultured on Murashige and Skoog medium containing 5 μm 2,4-dichlorophenoxyacetic acid. Shoot buds were produced when the callus was subcultured on a medium containing a cytokinin or a cytokinin and 1-naphthaleneacetic acid (NAA). The maximum number of shoots was formed on the medium containing thidiazuron (1 μM), or benzylaminopurine (5 μM) and NAA (1 μM). Shoots were multiplied by forced axillary branching and rooted in vitro. Endosperm-derived plants were established in soil. Each of the ten plants examined cytologically was triploid (3 n=42). Received:17 February 1999 / Revision received: 4 May 1999 / Accepted: 19 May 1999     

Production of triploid plants from endosperm cultures of Phlox drummondii

Triploid plants of ornamental Phlox drummondii Hook. were raised from cultures of endosperm excised from immature fruits having zygotic embryo at early dicotyledonous stage. Endosperm tissue was firstly cultured with the embryo on the Murashige and Skoog’s (MS) medium supplemented with 5 μM 6-benzylaminopurine (BAP) + 10 μM α-napthaleneacetic acid (NAA) for 7 d and recultured after the embryo was removed. A friable callus appeared two weeks after removal of the embryo and it became compact callus mass in another three weeks. Upon transfer of this 5-week-old callus to the MS medium with 10 μM BAP + 2.5 μM indole-3-acetic acid (IAA), maximum percentage of green nodular shoot buds appeared from which regenerated dwarf shoots. Elongation of the dwarf shoots, however, required transfer of the individual dwarf shoots excised from the callus on the fresh medium and best results achieved on medium with low concentration of IAA (0.5 μM) in presence of 10 μM BAP. The shoots were then rooted in vitro and plants subsequently established in pots containing soil. Over 70 % of plants were triploid with a chromosome number of 2n=3x=21. Size of stem, leaves, flowers, pollen, and stomata of these triploid plants were higher and the plants were more vigorous as compared to naturally occurring diploid plants. In particular, flowers showed bright colour with enlarged central eye adding to their ornamental value.     

Production of triploid plants from the immature and mature endosperm cultures of rice

Summary Triploid plants were induced to regenerate from immature and mature endosperm cultures of three cultivars of rice. The plants were subsequently established in the soil. The significance of the induction of genetic variability in the germplasm of rice is discussed.     

Induction of triploid Citrus plants from endosperm calli in vitro

Summary Triploid hybrid Citrus plants were regenerated by somatic embryogenesis in vitro from endosperm derived calli. A sequence of media formulations was used to induce and support proliferation of primary callus from endosperm, to induce embryogenesis from primary callus, and to allow embryo development leading to viable plantlets. Calli were induced from cellular endosperm of Citrus sinensis (sweet orange), C. Xparadisi (grapefruit), and C. grandis (pummelo) excised 12–14 weeks post-anthesis. Induction of embryogenesis from sweet orange and pummelo primary calli required gibberellic acid and double mineral nutrient concentrations. Embryogenesis was not induced from grapefruit calli in these experiments. Only sweet orange embryos developed sufficiently to allow plant regeneration. Triploid axillary buds were minigrafted onto etiolated diploid rootstock seedlings in vitro in order to transfer triploid regenerants to soil and the external environment. Triploidy (2n = 3x = 27) was observed consistently in all phases of regeneration and in recovered plants. These results demonstrate that triploid hybrid plant recovery from Citrus endosperm can overcome barriers to sexual hybridization resulting from apomixis.Florida Agricultural Experiment Station Journal Series No. R-00627     

Somatic embryogenesis and regeneration of triploid plants in endosperm cultures of Acacia nilotica

Immature endosperm of Acacia nilotica formed a nodular callus on MS medium supplemented with 2,4-D, BAP and CH. In the third passage on this medium, in the dark, the callus differentiated somatic embryos. The embryos germinated on MS only after 15 d pre-treatment on modified MS medium in which major salts were replaced by those of major salts of B₅ medium and supplemented with glutamine, CH and CW. Triploid nature of the somatic embryos was confirmed by Feulgen cytophotometry.Abbreviations ABA abscisic acid - AC activated charcoal - BAP 6-benzylaminopurine - B₅ Gamborg et al. (1968) medium - CH casein hydrolysate - CW coconut water - d days - MS Murashige and Skoog (1962) medium - PEG 4000 polyethylene glycol - MW 3500–4000 - 2,4-D 2,4-dichlorophenoxyacetic acid     

Experimental induction of triploid plants ofPhysalis through anther culture

Summary Anther culture ofPhysalis minima L. (2 n=48) was successful and embryos and plantlets could be obtained 5–6 weeks after culture. MS medium containing CM was essential for pollen division. 16.7% of the cultured anthers produced plantlets. All the pollen plantlets were triploid (3 n=72). Regeneration of multiple shoot buds was obtained from cultured leaf and stem expiants of pollen plantlets.     

A model of triploid endosperm evolution driven by parent-offspring conflict

The parental investment in angiosperms comprises the endosperm, a nutrient reserve that is used during seed development. The endosperm contains genes from both parents. The most common endosperm form is the 3n Polygonum -type with more maternal genetic influence than paternal, i.e. with two maternal nuclei and one paternal nucleus. The evolutionary original state is thought to be a diploid endosperm with equal influence of the parents. We focus on the evolution of the triploid endosperm and show that a gene for triploid endosperm would have an initial advantage in a population of diploid endosperm type plants, and increase to fixation. We assume that endosperm amount is controlled by endosperm genes. Then a gene causing triploid endosperm will increase the influence of the mother plant on parental investment. The production of endosperm with two copies of the maternal genes will modify the inheritance of endosperm amount and cause an increased production of seeds.     

Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut,Juglans regia L. cv Manregian

Plants were regenerated by somatic embryogenesis from endosperm tissue of open-pollinated seeds of Juglans regia L. cv Manregian. These plants were obtained by growing endosperm tissue on media similar to those used for plant regeneration from walnut cotyledons (Tulecke and McGranahan 1985). The plants appear morphologically uniform and have a triploid chromosome number of 3n=48. Nine plants have been grown to a young sapling stage in soil. This embryogenic line from endosperm has been maintained in culture for two years by the process of repetitive somatic embryogenesis.     

Production of aneuploid melon plants following in vitro culture of seeds from a triploid x diploid cross

Aneuploid melon plants (Cucumis melo L.) were obtained from in vitro cultured seed, which were produced by crossing triploid (3x=36) x diploid (2x=24) plants. Twenty-six fruits were obtained from pollination of 29 bisexual flowers of triploid plants. Seeds were collected from the fruits at 2, 3, 4, 5 and >7 weeks after pollination and germinated in vitro on Murashige & Skoogs (MS) medium. Embryos developed from 0.6 to 1.6% of the cultured seeds after three weeks in culture. Shoots developed from 0 to 47% of embryos after transfer to half-strength MS medium. Some (0 to 50%) of elongated shoots that rooted were subcultured on the same medium. Five rooted plantlets were obtained through culture of 5,353 seeds. Four of the plants were aneuploid, with chromosome numbers of 27, 35, 45 and 46, respectively, and the one was tetraploid (4x=48).     

Non-random chromosome arrangement in triploid endosperm nuclei

The endosperm is at the center of successful seed formation in flowering plants. Being itself a product of fertilization, it is devoted to nourish the developing embryo and typically possesses a triploid genome consisting of two maternal and one paternal genome complement. Interestingly, endosperm development is controlled by epigenetic mechanisms conferring parent-of-origin-dependent effects that influence seed development. In the model plant Arabidopsis thaliana, we have previously described an endosperm-specific heterochromatin fraction, which increases with higher maternal, but not paternal, genome dosage. Here, we report a detailed analysis of chromosomal arrangement and association frequency in endosperm nuclei. We found that centromeric FISH signals in isolated nuclei show a planar alignment that may results from a semi-rigid, connective structure between chromosomes. Importantly, we found frequent pairwise association of centromeres, chromosomal segments, and entire arms of chromosomes in 3C endosperm nuclei. These associations deviate from random expectations predicted by numerical simulations. Therefore, we suggest a non-random chromosomal organization in the triploid nuclei of Arabidopsis endosperm. This contrasts with the prevailing random arrangement of chromosome territories in somatic nuclei. Based on observations on a series of nuclei with varying parental genome ratios, we propose a model where chromosomes associate pairwise involving one maternal and one paternal complement. The functional implications of this predicted chromosomal arrangement are discussed.     

Mapping quantitative trait loci underlying triploid endosperm traits

Endosperm, which is derived from two polar nuclei fusing with one sperm, is a triploid tissue in cereals. Endosperm tissue determines the grain quality of cereals. Improving grain quality is one of the important breeding objectives in cereals. However, current statistical methods for mapping quantitative trait loci (QTL) under diploid genetic control have not been effective for dealing with endosperm traits because of the complexity of their triploid inheritance. In this paper, we derive for the first time the conditional probabilities of F(3) endosperm QTL genotypes given different flanking marker genotypes in F(2) plants. Using these probabilities, we develop a multiple linear regression method implemented via the iteratively reweighted least-squares (IRWLS) algorithm and a maximum likelihood method (ML) implemented via the expectation-maximization (EM) algorithm to map QTL underlying endosperm traits. We use the mean value of endosperm traits of F(3) seeds as the dependent variable and the expectations of genotypic indicators for additive and dominance effect of a putative QTL flanked by a pair of markers as independent variables for IRWLS mapping. However, if an endosperm trait is measured quantitatively using a single endosperm sample, the ML mapping method can be used to separate the two dominance effects. Efficiency of the methods is verified through extensive Monte Carlo simulation studies. Results of simulation show that the proposed methods provide accurate estimates of both the QTL effects and locations with very high statistical power. With these methods, we are now ready to map endosperm traits, as we can for regular quantitative trait under diploid control.     

应用胚挽救技术获得三倍体柑橘植株

以3个柑橘异源四倍体体细胞杂种(即四倍体粗柠檬与哈姆林甜橙体细胞杂种,简称HR;酸柚与粗柠檬体细胞杂种,简称SR;墨西哥来檬与伏令夏甜橙体细胞杂种,简称KV)为父本,分别与二倍体单胚性沙田柚进行有性杂交,在有性胚还没有完全败育以前,通过胚抢救技术进行三倍体植株培养(以四季柚花粉亲本为对照)。结果表明,处理的杂交组合直接成苗率极显著低于对照(P0.01),其中又以沙田柚×HR组合成苗率相对较高,达10.1%;较适合瘪籽沙田柚幼胚离体培养的培养基是MT+GA31.0mg/L+蜂皇浆200mg/L+水解乳蛋白250mg/L+蔗糖30g/L+琼脂0.7%;流式细胞分析仪检测结果显示,本试验成功获得了柑橘三倍体植株。     

Aneuploid plants derived from crosses with triploid grapes through immature seed culture and subsequent embryo culture

Through immature seed culture and subsequent embryo culture, aneuploid plants were derived from various crosses among 184 different triploid hybrid grape vines. In self-pollinations of the 184 vines, 0 to 1.6% of flowers produced immature seeds. In 16 reciprocal crosses between diploid and triploid and between tetraploid and triploid grapes, 0 to 23.0% of flowers produced immature seeds. The immature seeds excised 30–50 days after pollination were cultured for three months on Nitsch and Nitsch medium supplemented with L-glutamine, L-serine, L-cysteine and casein hydrolysate. Embryos developed within the cultured immature seeds were subcultured onto germination medium consisting of MS medium with 1 μM BA. Thirty-four of 137 embryos from 458 immature seeds germinated. Five of the 34 embryos grew normally. The five recovered plants were aneuploids with chromosome numbers from 51 to 59. The rates of embryo and plant recovery were different in different crosses with triploid grapes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Anther culture of papaya (Carica papaya L.)

Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid plantlets and pollen-derived embryoids were obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.Abbreviations MS Murashige and Skoog (1962) - MAA naphthaleneacetic acid - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid     

An efficient protocol for the production of triploid plants from endosperm callus of neem,Azadirachta indica A. Juss

Triploid plants of neem were obtained by immature endosperm culture. Immature seeds, at the early dicotyledonous stage of embryo development, is the best explant to raise endosperm callus on MS + NAA (5 mumol/L) + BAP (2 mumol/L) + CH (500 mg L-1). Maximum shoot bud differentiation from the endosperm callus occurred on MS + 5 mumol/L BAP. Shoots were multiplied by forced axillary branching and rooted in vitro. The plants were established in soil. Over 66% of the plants were triploid with chromosome number 2n = 3x = 36. A characteristic feature of the shoots of endosperm origin is the presence of a large number of multi-cellular glands.     

Production of doubled-haploid plants from tritordeum anther culture

The effects of different media and cold pretreatment of spikes on the androgenic response and regeneration capacity from anther culture of tritordeum was studied. L5 medium gave the highest frequency of anther response. The frequency of cultures regenerating green or albino plantlets was not affected by the composition of the medium tested. Cold pretreatment of the spikes significantly increased the frequency of anther response and also the percentage of cultures giving albino plantlets. A mean of four green plants was obtained per 100 subcultured calli/embryos. The percentage of spontaneous chromosome doubling was only 1%. The addition of colchicine at 0.02% to the induction medium significantly increased the frequency of doubled haploids regenerated without any effect on regeneration capacity. This technique proved more efficient than a conventional chromosome-doubling method.     

Statistical model for characterizing epistatic control of triploid endosperm triggered by maternal and offspring QTLs

To study the effects of maternal and endosperm quantitative trait locus (QTL) interaction on endosperm development, we derive a two-stage hierarchical statistical model within the maximum-likelihood context, implemented with an expectation-maximization algorithm. A model incorporating both maternal and offspring marker information can improve the accuracy and precision of genetic mapping. Extensive simulations under different sampling strategies, heritability levels and gene action modes were performed to investigate the statistical properties of the model. The QTL location and parameters are better estimated when two QTLs are located at different intervals than when they are located at the same interval. Also, the additive effect of the offspring QTLs is better estimated than the additive effect of the maternal QTLs. The implications of our model for agricultural and evolutionary genetic research are discussed.     

The triploid endosperm genome of Arabidopsis adopts a peculiar, parental-dosage-dependent chromatin organization

The endosperm is a seed tissue unique to flowering plants. Due to its central role in nourishing and protecting the embryo, endosperm development is subject to parental conflicts and adaptive processes, which led to the evolution of parent-of-origin-dependent gene regulation. The role of higher-order chromatin organization in regulating the endosperm genome was long ignored due to technical hindrance. We developed a combination of approaches to analyze nuclear structure and chromatin organization in Arabidopsis thaliana endosperm. Endosperm nuclei showed a less condensed chromatin than other types of nuclei and a peculiar heterochromatin organization, with smaller chromocenters and additional heterochromatic foci interspersed in euchromatin. This is accompanied by a redistribution of the heterochromatin mark H3K9me1 from chromocenters toward euchromatin and interspersed heterochromatin. Thus, endosperm nuclei have a specific nuclear architecture and organization, which we interpret as a relaxed chromocenter-loop model. The analysis of endosperm with altered parental genome dosage indicated that the additional heterochromatin may be predominantly of maternal origin, suggesting differential regulation of maternal and paternal genomes, possibly linked to genome dosage regulation.     

Lateral bud culture of papaya (Carica papaya L.) for clonal propagation

Lateral buds may be preferred to shoot tips for in vitro propagation of papaya because of its unbranched nature. Proliferating shoot cultures from lateral buds appeared extremely compact with shortened internodes and leaf lamina of the cytokinin level (BAP 2 M) reported for multiple shoot production from shoot tips. ZEA (4 M) and 2iP (8 M) although reduced the proliferation rate, resulted in better growth of the shoot from lateral bud. Rooting was observed with IBA 20 M but plantlets so produced remained stunted.     

Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.