In vitro tetraploid induction and generation of tetraploids from mixoploids in Astragalus membranaceus

This paper describes an efficient colchicine-mediated technique for the in vitro induction of tetraploids in Astragalus membranaceus and its confirmation by flow cytometry. Buds immersed in 0.2% colchicine solution for 36 h prior to culture induced as high as 35.3% tetraploid plants. Colchicine-induced tetraploids remained stable after 6 months in soil. Leaf characteristics of diploids and tetraploids in A. membranaceus were compared. It was determined that leaf sizes of glasshouse-grown plants and stomatal sizes of both in vitro and glasshouse-grown plants were suitable parameters for identifying putative tetraploids in A. membranaceus. As well as generating tetraploids, this technique generated mixoploids in A. membranaceus. Calli derived from mixoploid leaves, were induced to form buds and shoots. Individual shoots were classed as diploid, mixoploid and tetraploid by flow cytometry. This callus-based technique can be employed when a genome-doubling agent generates mixoploids but no tetraploid.     

Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.)

The tips of cotyledon-stage seedlings of three crape myrtle cultivars (“Zi Wei”, “Hong Wei” and “Yin Wei”) were treated with colchicine. Various concentrations of colchicine and different treatment durations were tested. Seedlings of “Zi Wei” treated with 0.5% colchicine for 72 h and seedlings of “Yin Wei” treated with colchicine (0.2% for 96 h, 0.5% for 48 h and 0.8% for 72 h) demonstrated high rates of mutation; “Hong Wei” showed a slightly lower rate. The highest rate of morphological variation was 54.17% and this was achieved when tips were treated with 0.5% colchicine for 72 h. Putative tetraploid plants were identified with morphological and cytological variations, such as larger and thicker leaves, darker green coloration, larger stomata, lower density of stomata across the lower leaf epidermis and increased numbers of chloroplasts per stomata guard cell. Flow cytometric analysis demonstrated that the nuclear DNA content of 15 of these putative tetraploids (of which 7 were “Zi Wei”, 5 were “Yin Wei”, and 3 were “Hong Wei”) was indeed doubled relative to that of control diploid plants. The chromosome number of the tetraploid plants was 2n = 4x = 96, while that of the control diploid plants was 2n = 2x = 48. Ornamental characteristics were also enhanced in the tetraploid plants, with increased diameter of the individual flowers and greater basal length of the petals and claws. Pollen diameter and the size of capsules and seeds were also significantly greater than those of diploid plants.     

Induction of tetraploidy in meristematically active seeds of Japanese barberry (Berberis thunbergii var. atropurpurea) through exposure to colchicine and oryzalin

Japanese barberry (Berberis thunbergii DC) is an invasive shrub, widely naturalized across the United States, whose numerous cultivars remain an important horticultural commodity. Maintaining this crop for the future necessitates the development of sterile clones. Exposure to the mitotic inhibitors colchicine and oryzalin is a traditional method for inducing tetraploidy in breeding lines as a precursor to creating sterile genotypes. Treatments utilized pre-germinated B. t. var. atropurpurea seeds with emerged radicles. Seeds were immersed in aqueous solutions of colchicine (.02%, .05%, .1% and .2%) and oryzalin (.002%, .005%, .01% and .02%) dissolved in 1% DMSO for 6, 12 and 24 h durations. Seedling ploidy level was determined via flow cytometry following 6 and 52 weeks of growth in the greenhouse. Both anti-mitotic chemicals proved effective at inducing tetraploidy and produced comparable efficiency rates. The survival rate of treated seeds decreased in response to both increased mitotic inhibitor concentration and longer exposure duration. While exposure to oryzalin produced greater seed mortality than colchicine, most seedlings that survived had altered ploidy levels. The most efficient oryzalin concentration was 0.002% with a rating of 28%, while the most efficient colchicine concentrations were in the range from 0.05% to 0.2%. Duration of exposure to mitotic inhibitor was not a significant factor over the range from 6 to 24 h. Reversion of tetraploid plants to the diploid state occurred at a low frequency following a dormancy period. Some tetraploid seedlings derived from exposure to both chemicals displayed foliar abnormalities including irregular leaf margins and mottled lamina. The primary advantage of colchicine was low seedling toxicity, while oryzalin was notable for its ability to induce tetraploidy at low concentrations.     

In situ induction of chromosome doubling in vine cacti (Cactaceae)

Vine cacti have economic potential as exotic fruits in semi-arid and arid lands due to their high water use efficiency. The goal of this study was to attain autopolyploid plants by applying the antimitotic agents colchicine and oryzalin on axillary vegetative buds and germinating seeds. The diploid Hylocereus monacanthus and the tetraploid H. megalanthus; the interspecific triploid hybrid S-75, were studied. The effects of different concentrations and exposure times on bud survival and germination rate were recorded. A negative effect on vegetative bud survival was observed in the triploid hybrid. An inhibitory effect on germinating seeds was species specific, being greater on H. monacanthus with oryzalin and on H. megalanthus with colchicine. Putative polyploids were identified by comparing stomatal density between donor plants and plants arising from treated buds or seeds. Flow cytometric analysis and chromosome counts confirmed polyploidization in 14 autotetraploid H. monacanthus, 1 autohexaploid S-75 hybrid, and 14 autooctaploid H. megalanthus. This is the first report of artificial autopolyploidization in species of vine cactus, providing valuable novel plant material for further breeding programs.     

Colchicine- and trifluralin-mediated polyploidization of Rosa multiflora Thunb. var. inermis and Rosa roxburghii f. normalis

Rosa multiflora Thunb. var. inermis and Rosa roxburghii f. normalis are two important diploid species for rose breeding. Interspecific crosses between diploid species and tetraploid rose cultivars result in triploids with generally decreased fertility. A most promising gene transfer strategy is the production of fertile tetraploid plants before mating with tetraploids. In our study, germinating seeds of R. multiflora and R. roxburghii were treated with colchicine, and cotyledon-stage seedlings of R. multiflora were treated with colchicine or trifluralin in order to obtain tetraploid plants. Also, various concentrations of antimitotic agents and different treatment durations were tested. The results showed the antimitotic agents affected the plant morphological characteristics (i.e. plant height, stem diameter) of R. multiflora and R. roxburghii. Twenty-three tetraploid plants were obtained from the germinating seed treatments (21 from R. multiflora and two from R. roxburghii), but no tetraploid plants were obtained from the seedling treatments. The highest rate of chromosome doubling (25.0%) was achieved when germinating seeds of R. multiflora were treated with 0.2% colchicine for 12 h. At the same time, marked morphological variation was observed and analyzed among tetraploid plants and their corresponding diploids of R. multiflora.     

Tetraploid muskmelon alters morphological characteristics and improves fruit quality

Tetraploid muskmelon plants were induced successfully from diploid Cucumis melo inbred M01-3 (2n = 24) by colchicine. The morphological characteristics and fruit qualities of diploid and tetraploid muskmelons were investigated. The results showed that the leaves and flowers of the tetraploid plants were markedly larger, the plants were obviously higher, and the stems were thicker than those of the diploid plants. Transmission electron microscope observation revealed that the numbers of chloroplast, granule and grana, and the length of chloroplast and granule of the tetraploid plant leaves were significantly more or longer than those of the diploid plants. The soluble solid, soluble sugar and vitamin C contents in the tetraploid fruit were distinctly higher than those in the diploid fruit. The weight of the tetraploid fruit was 30% heavier than that of the diploid fruit. And the seeds from the tetraploid plants were broader and thicker than those from the diploid plants. Together, our results suggest that the tetraploid muskmelon exhibited better agronomical characteristics than the diploid muskmelon and the tetraploid muskmelon could be used as the improved variety and a potential germplasm for the development of triploid fruit.     

In vitro induced tetraploid of Dendranthema nankingense (Nakai) Tzvel. shows an improved level of abiotic stress tolerance

The tetraploid of Dendranthema nankingense (Nakai) Tzvel. was induced by the colchicine treatment using nodal segments. Ploidy level was determined by an analysis of flow cytometry and chromosome counting. The morphological characteristics such as the stomata, leaves, flowers and pollen grains of the tetraploid were significantly larger than those of the diploid. The tolerance responses of the diploid and tetraploid were compared under the imposition of heat, cold, drought and salinity stress. Semi-lethal temperatures suggest that cold tolerance is improved by polyploidization, but the heat tolerance is reduced. Under drought and salt stress, the activity of peroxidase (POD) and relative water content (RWC) in the tetraploid were higher than those in the diploid. Accordingly its malondialdehyde (MDA) content maintained a lower level. The content of chlorophyll (a + b) in the tetraploid was higher, and decrease of its content was postponed in tetraploid compared with the diploid under salt stress. It suggested that polyploidization could alleviate oxidative stress, maintain good water balance and higher chlorophyll (a + b) content, thereby enhanced the drought and salt tolerance in colchicine induced tetraploid.     

Ploidy variation of hardy kiwifruit (Actinidia arguta) resources and geographic distribution in Japan

Survey on the wild genetic resources of hardy kiwifruit (Actinidia arguta) in Japan was conducted to determine the ploidy variation and its geographic distribution. Among the 127 wild plants collected from different geographic locations, 15 plants were diploid, 87 were tetraploid, and 22 were hexaploid. Additionally, 2 plants were heptaploid and one plant was octaploid. The tetraploid plants were distributed all over the country, whereas the diploid and hexaploid plants were geographically localized, in the warm Pacific hill areas of the south western part and in the deep-snow region of the mid-northern part of Honshu, respectively. The diploid plants could be clearly distinguished from other plants with ploidy variation by the morphological characteristics of the leaf and fruit. Hexaploid plants showed a relatively larger L/D ratio of the leaf blade, a greenish petiole, and pubescence on the petiole and lower leaf vein, whereas the tetraploid plants exhibited a reddish petiole and callose hairs on the vein of the lower leaf surface. Fruit shape of the tetraploid plants varied largely, from round to ellipsoidal, whereas that of the hexaploid plants was mostly ellipsoidal. These results indicate that the hexaploid plants of A. arguta as well as the diploid and tetraploid ones, naturally grow in a certain size of population in the restricted region of Japan.     

Effect of Glomus mosseae and Entrophospora colombiana on plant growth,production, and fruit quality of ‘Maradol’ papaya (Carica papaya L.)

The effect of inoculating ‘Maradol’ papaya plants with arbuscular mycorrhizal fungi (AMF) Glomus mosseae (GM) and Entrophospora colombiana (EC) was assessed. The results showed that both mycorrhizae species increased the number of fruits and yield in papaya plants by 41.9 and 105.2% for GM and 22.1 and 44.1% for EC, respectively, with respect to control plants. GM significantly increased plant height. Sugar content, firmness, color (°Hue), and ripening process of mycorrhized plant fruits were similar to those of the control. Weight loss of mycorrhized plant fruits was considerably less than that of the control. Inoculation of papaya with AMF is recommended, particularly with GM since it increases yield, and fruit weight (45.1%), furthermore, it reduced fruit weight loss during ripening.     

Plant characteristics for distinction of diploid,triploid and tetraoloid lettuce

After colchicine treatments of lettuce cultivars, tetraploid plants have been obtained. Crosses between 4x and 2x plants resulted in 3x plants. Between these 3 ploidy levels differences occurred in many plant characteristics which can be used as selection criteria. The number of plastids per stoma is suitable for distinction between the 3 ploidy levels. Stomatal length, size of flower, pollen grain length, seed length and seed weight can be used for distinguishing 4x and 2x plants. The percentage of stained pollen grains and the seed production per plant can be used for distinguishing 3x and 2x plants.     

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).     

Plant growth regulators and flowering of Brunonia and Calandrinia sp.

Brunonia australis R. Br (Goodeniaceae) and Calandrinia (Portulacaceae), native to Australia, are potential new flowering potted plants. This research investigated the role of daylength and growth regulators, Gibberellic acid (GA₃) and paclobutrazol (Pac), to control vegetative growth, peduncle elongation and flowering of Brunonia and Calandrinia. Plants were grown under long days (16 h), short days (11 h) and 8 weeks under short day then transferred to long day (SDLDs). Plants in each daylength were treated with GA₃, Pac, and GA₃+ Pac. GA₃ was applied as 10 μL drop of 500 mg L⁻¹ concentration to the newest mature leaf. A single application of Pac was applied as a soil drench at 0.25 mg a.i. dose per plant. Both Brunonia and Calandrinia flowered earlier in long days but still flowered in short days, so both can be classified as facultative LD plants. Brunonia under SDLDs were more vigorous and attractive than plants under LDs while still being more compact than plants under SDs. In Brunonia, GA₃ promoted earlier flowering and increased the number of inflorescences under SDs. Pac at 0.25 mg a.i. per plant applied alone or in combination with GA₃ had extended flower development in Brunonia, and resulted in a reduced number of inflorescences per plant compared to the control plants. Vegetative growth of Calandrinia was similar under LDs, SDs and SDLDs, whereas GA₃ application increased plant size. Pac-treated Calandrinia looked compact and attractive, and Pac application did not affect time to flower and flower number.     

The culture of isolated microspores of ornamental kale (Brassica oleracea var. acephala) and the importance of genotype to embryo regeneration

The culture of isolated microspores of kale (Brassica oleracea var. acephala) was studied including the importance of genotype to embryo regeneration, medium composition chiefly the sucrose concentration and the use of colchicine, simultaneously medium renovation. It was initiated using 29 different genotypes as donor plants. Embryos were induced from six of the kale genotypes and these corresponded to the more out-bred genotypes. Embryogenesis was achieved using four different combinations of culture media: (a) microspores initially cultured in NLN medium supplemented with 13% (w/v) sucrose (NLN-13) for 48 h, followed by transfer to fresh NLN-13 medium; (b) microspores cultured for 48 h in NLN-13 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium; (c) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-16 medium; (d) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium. The embryos obtained from four of the genotypes developed into plantlets and these regenerated plants have been successfully transplanted to soil.     

Molecular analysis revealed autotetraploid,diploid and tetraploid cybrid plants regenerated from an interspecific somatic fusion in Citrus

More than 150 plants were regenerated from our previous somatic hybridization between embryogenic callus line of Page tangelo (Citrus reticulata Blanco × C. paradisi Macf) and mesophyll protoplasts of rough lemon (C. jambhiri Lush) mediated by electrofusion. Preliminary screening showed that 78% of these plants were tetraploids while the rest were diploids morphologically resembling the leaf parent (rough lemon). Herein, eight plants (six tetraploids and two diploids) were selected and further analyzed by flow cytometry, simple sequence repeat (SSR), mitochondria (mt) RFLP and chloroplast (cp) SSR techniques. The results showed that four of these six tetraploids were somatic hybrids, one tetraploid was autotetraploid of Page tangelo, and the remaining one tetraploid was cybrid with nuclear and cpDNA from rough lemon and mtDNA from Page tangelo; the two diploids were verified being cybrids with nuclear DNA from rough lemon and mtDNA from Page tangelo, cpDNA was randomly inherited. The regeneration mechanism of these novel cybrids was discussed.     

High frequency plantlet regeneration from callus and artificial seed production of rock plant Pogonatherum paniceum (Lam.) Hack. (Poaceae)

Pogonatherum paniceum (Lam.) Hack. is a rock plant with good potential for vegetative recovery on naked lands. A high frequency in vitro regeneration system was developed for P. paniceum. Calli were induced from explants of mature seeds, seedlings, young leaves, and stem segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 2.0 mg L⁻¹ α-naphthalene acetic acid (NAA) and 0.2 mg L⁻¹ 6-benzylaminopurine (BAP). High induction rates (59.57%) and regeneration rates (100%) were obtained from mature seed explants; calli were sub-cultured for over 2 years and still retained a high regenerative capacity. One seed explant resulted in 69,997 plants in 1 year. Shoot buds derived from calli were used for encapsulation in liquid MS medium containing 3% sucrose and two different alginate matrices (3% sodium alginate (w/v) + MS medium containing 3% sucrose and 3% sodium alginate + 1% activated carbon (w/v) + MS medium containing 3% sucrose) with a 20-min exposure to 2% CaCl₂ and 0.3% bavistin (w/v). The capsule with 3.0% sodium alginate (w/v) and 1% activated carbon (w/v) showed a higher conversion rate (61.58%) and stronger plantlets under non-aseptic conditions. These systems are useful for the rapid clonal propagation and dissemination of artificial seed material of P. paniceum for eco-recovery.     

Transpiration,photosynthetic responses,tissue water relations and dry mass partitioning in Callistemon plants during drought conditions

Callistemon is an Australian species used as ornamental plant in Mediterranean regions. The objective of this research was to analyse the ability of Callistemon to overcome water deficit in terms of adjusting its physiology and morphology. Potted Callistemon laevis Anon plants were grown in controlled environment and subjected to drought stress by reducing irrigation water by 40% compared to the control (irrigated to container capacity). The drought stress produced the smallest plants throughout the experiment. After three months of drought, the leaf area, number of leaves and root volume decreased, while root/shoot ratio and root density increased. The higher root hydraulic resistance in stressed plants caused decreases in leaf and stem water potentials resulting in lower stomatal conductance and indicating that water flow through the roots is a factor that strongly influences shoot water relations. The water stress affected transpiration (63% reduction compared with the control). The consistent decrease in g_s suggested an adaptative efficient stomatal control of transpiration by this species, resulting in a higher intrinsic water use efficiency (P_n/g_s) in drought conditions, increasing as the experimental time progressed. This was accompanied by an improvement in water use efficiency of production to maintain the leaf water status. In addition, water stress induced an active osmotic adjustment and led to decreases in leaf tissue elasticity in order to maintain turgor. Therefore, the water deficit produced changes in plant water relations, gas exchange and growth in an adaptation process which could promote the faster establishment of this species in gardens or landscaping projects in Mediterranean conditions.     

Rapid regeneration of plants of Dendrobium lituiflorum Lindl. (Orchidaceae) by using banana extract

Effects of banana extract (BE) and 6-benzylaminopurine (BAP) were evaluated on asymbiotic seed germination and an early differentiation of protocorms and plant regeneration of Dendrobium lituiflorum Lindl. High percentage germination was achieved by culturing seeds on modified Knudson C medium supplemented with 10% (v/v) BE. Rapid regeneration was observed within 60 days of culture on 10% (v/v) BE supplemented KC medium where maximum percentage propagules showed development of leaves and root formation. Propagules on BAP supplemented KC medium showed no further development beyond one leaf stage. In another experiment, culture of shoots on 12.5% (v/v) BE supplemented KC medium led to multiplication, shoot elongation as well as vigorous rooting. Shoots cultured on 10 μM BAP supplemented MS medium showed maximum multiplication but these were stunted. Plants with well expanded deep green leaves and elongated roots from BE media were first hardened in vitro followed by ex vitro hardening on cocopeat:perlite (9:1) in the greenhouse conditions and exhibited 90% survival. The study emphasizes the role of BE as a natural additive at different stages of development from seed germination to plant regeneration.     

寒富苹果叶片离体再生及四倍体诱导

为建立寒富苹果高效的离体再生体系和多倍体诱导体系,以试管苗叶片为外植体,研究了培养基中激素对寒富叶片再生芽的影响及适宜的四倍体诱导方法。结果表明,当培养基中BA质量浓度为0.5mg/L时,再生频率最高达35.7%;当培养基中BA质量浓度为2.5mg/L时,再生频率超过90%,平均再生芽数在4以上。在培养基中附加1.0mg/LTDZ,再生频率达100%,平均再生芽数达19.47。以附加15、30、60、120mg/L秋水仙素的液体再生培养基处理叶片5d,各个质量浓度处理均诱导出四倍体植株,诱变率在5.3%～22.2%之间;寒富苹果叶片在附加50mg/L秋水仙素固体再生培养基上处理5d亦获得了四倍体植株。研究结果表明寒富苹果叶片具有极强的不定芽再生能力,叶片再生过程中进行秋水仙素处理是获得其四倍体的一个有效途径。     

The influence of planting density on the production of ‘Goldfinger’ (Musa spp., AAAB) in the subtropics

‘Goldfinger’, a tetraploid banana produced from the Fundación Hondureña de Investigación Agrícola (FHIA) breeding program, was released to the Australian industry in 1995. It was promoted as an apple-flavoured dessert banana with resistance to Fusarium wilt race 1 and subtropical race 4, as well as resistance to black and yellow Sigatoka (Mycosphaerella fijiensis and M. musicola, respectively). This study was initiated to provide agronomic information to the banana industry, which was under threat from Fusarium wilt, on a new cultivar which could replace ‘Williams’ (AAA, Cavendish subgroup) or ‘Lady Finger’ (AAB, Pome subgroup) in those areas affected by Fusarium wilt. Also few studies had reported on the production characteristics of the new tetraploid hybrids, especially from subtropical areas, and therefore two field sites, one a steep-land farm and the other a level, more productive site, were selected for planting density and spatial arrangement treatments. The optimum density in terms of commercial production, taking into account bunch weight, finger size, length of the production cycle, plant height and ease of management, was 1680 plants/ha on the steep-land site where plants were planted in single rows with 2.5 m × 2.5 m spacings. However on the level site a double-row triangular layout with inter-row distances of 4.5 m to allow vehicular access (1724 plants/ha) gave the best results. With this arrangement plants were in an alternate, triangular arrangement along a row and a spacing of 1.5 m between plants at the points of each triangle and between each block of triangles.