Efficient regeneration of plants independent of exogeneous growth regulators in bell pepper (Capsicum annumm L.)

Summary Organogenesis of shoots of bell pepper (Capsicum annuum L.) was achieved in fourteen cultivars on Murashige and Skoog's medium (MS medium) supplemented only with 0.4% (w/v) Gellan gum (pH 5.8). Mature seeds of cv. Shinsakigake-2 were sown on filter paper that had been wetted with sterilized water and precultured for zero to five days in under 16 hr of light per day at 25 °C. Explants, consisting of the proximal part of the hypocotyl and the radicle, were excised from the seeds and formed adventitious buds around the cut surfaces of elongated hypocotyls after four weeks of culture. When explants were subcultured on MS medium, 57% of the explants that had produced adventitious buds extended shoots after an additional three weeks of culture. Shoots were rooted on MS medium after two further weeks of culture. Chromosome numbers of all 30 regenerated plants that weexamined were normal (2n=24). The morphology of the mature plants was also normal and they set normally shaped fruits with mature seeds. Regenerated whole plants were also obtained in the case of 13 other cultivars by applying this simple procedure.Abbreviations MS Murashige and Skoog     

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.     

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.     

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.     

Extensive citrus triploid hybrid production by 2x?×?4x sexual hybridizations and parent-effect on the length of the juvenile phase

The citrus fresh market demands the production of seedless citrus fruits, as seedy fruits are not accepted by consumers. The recovery of triploid plants has proven to be the most promising approach to achieve this goal, since triploids have very low fertility, are generally seedless and do not induce seeds in other cultivars by cross pollination. Triploid plants can be recovered by 2x?×?4x sexual hybridization. In this work, we present an effective methodology to recover triploid plants from 2x?×?4x hybridizations based on in vitro embryo rescue, ploidy level analysis by flow cytometry and genetic origin of triploid plants. The pollen viability of diploid and tetraploid citrus genotypes was analyzed by comparing the pollen germination rate in vitro. The pollen viability of tetraploid (doubled-diploid) genotypes is generally reduced but sufficient for successful pollination. Triploid embryos were identified in normal and undeveloped seeds that did not germinate under greenhouse conditions. The influence of parents and environmental conditions on obtaining triploid plants was analyzed and a strong interaction was noted between the parents and environmental conditions. The parental effect on the length of the juvenile phase was also demonstrated through observations of a large number of progeny over the last 15?years. The juvenile phase length of the triploid hybrids obtained with 'Fortune' mandarin as female parent and tetraploid 'Orlando' tangelo as male parent was shorter than the juvenile phase obtained with a clementine as female parent and tetraploids of 'Nova', 'W. Leaf' and 'Pineapple' male parents. Key message Effective methodology to recover citrus triploid plants from 2x?×?4x sexual hybridizations and the parental effect on the length of the juvenile phase.     

Recovery of cassava (Manihot esculenta Crantz) plants from culture of immature zygotic embryos

An embryo culture protocol using immature cassava seeds has been developed to enhance successful seed germination and reduce time for population establishment. Embryonic axes were excised from seeds 40 days after pollination and placed on 1/3 MS medium supplemented with growth factors. Fruits were either air-dried at 20 °C to aid dehiscence, or dissected immediately after harvest. Culture of embryonic axes from seeds obtained from mature fruits (90 days after pollination) served as control. Average percent germination and plantlet recovery rate were higher for embryos cultured from non air-dried immature seeds than from air-dried immature seeds. Immature seeds that were air-dried before germination had ≥50% reduction in germination rate and ≥75% reduction in plantlet recovery rate, indicating that cassava immature zygotic embryos are susceptible to osmotic pressure changes. Genotypic effects were observed in shoot elongation, formation of internodes, and vigor of cultures from both mature and immature seeds. The high percentage of plants recovered from immature seeds through embryo culture opens up opportunities for genetic stock development in cassava that has been previously unexplored. This revised version was published online in June 2006 with corrections to the Cover Date.     

以异源四倍体体细胞杂种为父本杂交培育三倍体柑桔植株的研究

以 3个柑桔原生质体融合而来的四倍体体细胞杂种为父本 ,与二倍体单胚性种柚子 (Citrusgrandis)以及单多胚混合型品种“华农本地早”桔 (C.reticulata)有性杂交 ,授粉后 90 d,发现种子干瘪 ,大部分种子的胚败育。将干瘪种子在 MT附加 1mg/L GA3 或 50 0 mg/L麦芽浸出物的培养基中 ,经培养抢救 ,有 2 5.6%的种子萌发成苗或继续进行胚的生长 ,后者进一步诱导能形成丛芽 ,经试管嫁接或诱导生根形成完整植株。共获得 6个组合 73棵完整植株 ,染色体数检查表明 ,2 0株为三倍体 (2 n=3x=2 7) ,32株为二倍体 (2 n=2 x=18) ,8株为非整倍体 ,其它 13株还有待于进一步检查。     

Triploid Citrus Plants Obtained from Crossing the Diploids with Allotetraploid Somatic Hybrids

Hybridizing the diploid monoembryonic pummelo (Citrus grandis) and polyembryonic tangerine (C. reticulata cv. Huanongbendizao) with allotetraploid somatic hybrids from protoplast fusion were conducted. Seeds of pollinated fruits were found to be abortive 90 days after pollination. The aborted seeds were then cultured on media of MT supplement with 1 mg/L GA3 or with 500 mg/L of malt extract. 25.6% of the seeds germinated or developed into embryoids: The entire plants were transplanted into soil after grafting shoots on the root-stocks of trifoliate orange in vitro, if the germinated embryos have poor roots or no root at all. A total of 73 plants from 6 different combinations were obtained, among which 20 were verified as triploids with 2n= 3x=27 chromosomes, 32 diploids 2n= 2x= 18, 8 a- neuploids and the rest 13 unconfirmed.     

In vitro propagation of threatened terrestrial orchid, Malaxis khasiana Soland ex. Swartz through immature seed culture

Rapid in vitro propagation of the terrestrial orchid, M. khasiana through immature seed culture was achieved. Immature seeds of 8-9 week after pollination (WAP) cultured on MS medium (2% sucrose) supplemented with 500 mgl(-1) casein-hydrolysate and 1 microM N6-benzyladenine (BA) exhibited germination of 75% seeds after 107 days of culture and subsequently supported the development of PLBs. Subsequent culture on MS medium enriched with 6 microM of indole-3-acetic acid (IAA), 18 microM each of BA and kinetin induced multiple shoots and plantlets. Transfer of PLBs to MS medium with 0.1% activated charcoal (AC) facilitated rapid proliferation of PLBs, while AC at 0.2% favored shoot bud induction and rhizome enlargement. The plantlets, developed on medium with IAA, BA and kinetin, after hardening in vitro for 8-10 weeks were planted in community pots and transferred to poly-house. The plantlets showed 65% survival under field conditions.     

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.     

Asymbiotic seed germination and in vitro seedling development of Cymbidium aloifolium (L.) Sw.: a multipurpose orchid

Cymbidium aloifolium is a multipurpose economically important epiphytic orchid grows on tree trunk in the primary forests. Its population in natural habitat is downsized due to different anthropogenic activities. A successful attempt was made for asymbiotic immature embryo culture and in vitro mass scale production of plantlets. For successful culture initiation seed pods of various developmental ages, various nutrient media, sucrose concentrations, different quality and quantity of plant growth regulators were surveyed. Immature embryos of 9 months after pollination was successfully germinated on MS medium containing sucrose (2%) (w/v) and α-naphthalene acetic acid (NAA) and benzyl adenine (BA) (3 and 6 μM respectively in combination) within 45 days of culture where 90% germination was recorded. The germinated seeds formed PLBs on the optimum germination medium within two passages. The protocorm like bodies (PLBs) differentiated into rooted plantlets within 3 weeks on regeneration medium containing sucrose (3%), casein-hydrolysate (0.1 gl^?1) and BA 3 μM. Amongst the three media studied, optimum regeneration was registered on MS medium where as many as 12 shoot buds developed per explants per subculture of 4 weeks duration. The well rooted plantlets of 6–7 cm long with 3–4 roots were hardened in vitro 3–4 weeks before they were transferred to potting mix. The potted plants were exposed to full sunlight periodically and watered at regular interval. About 70–80% transplants survived after 2 months of potting.     

Direct plantlet regeneration from the tuber of Stachys sieboldii

Development of an efficient in vitro plantlet regeneration system from tubers of Stachys sieboldii (Miq.), a traditional Chinese medicinal plant and vegetable, is described. Adventitious shoots with an average of 9.1 (after 4 weeks of incubation) shoots per explant were obtained from 60.4% of tuber explants cultured on Murashige and Skoog (MS) medium containing 1.0 mg l^–1 thidiazuron (TDZ), and after another 4 weeks of incubation on growth regulator free MS medium, 33.5 shoots per explant were achieved. Subcultures showed results that were similar to those of the initial culture. Excised shoots were rooted both on MS and on half strength MS medium with a frequency of about 95%. Excised shoots were also rooted ex vitro by directly planting them into cups containing sand. Regenerated plants with well developed shoots and roots were successfully transplanted to soil, after which they developed free of abnormalities.     

Micropropagation of Alnus cordata (Loisel.) Loisel.

Procedures were developed for micropropagation of Alnus cordata through in vitro axillary shoot multiplication of axillary bud explants cultured in Murashige & Skoog (MS) medium. Establishment of cultures from plants grown in the field was very difficult due to bacterial contamination and phenolic oxidation in explants causing severe browning. Explants were first cultured on an MS medium containing 4.4 M 6-benzyladenine and 87.6 mM sucrose (initiation medium) for 7 days and then transferred to an MS medium containing 1.1 M 6-benzyladenine and 333 mM glucose (multiplication medium) for a further 20–25 days. It was necessary to transfer cultures from initiation medium to multiplication medium after 7 days to minimize excessive callus growth, abnormally thick and brittle leaves, inhibition of shoot elongation, and senescence. Shoot multiplication comparable to the above method was achieved by culture of axillary bud explants in MS medium supplemented with 1.1–4.4 M 6-benzyladenine and 333 mM glucose 4–5 weeks after culture. Shoots rooted in MS medium (1/2 x macro-nutrients) supplemented with 1.2–4.9 M indolebutyric acid. Also, 98% rooting was achieved when cultures were treated with 625 mgl^-1 indolebutyric acid for 24 h at the end of the shoot production stage and rooted in vivo as mini-cuttings. Plantlets established well in soil.     

Agrobacterium-mediated transformation and regeneration of cotton plants

Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and β-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assay. Shoots regenerated from excised shoot meristems or their halves were cultured for 4–6 weeks to obtain rooted plants, which then produced fully-developed plants and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with 50 mg/l kanamycin. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 462–467. The text was submitted by the authors in English.     

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

In vitro embryo rescue culture of F<Subscript>1</Subscript> progenies from crosses between diploid and tetraploid grape varieties

The purpose of this study was to understand factors affecting in vitro embryo rescue culture from hybrids between diploid and tetraploid varieties of grape in creation new triploid germplasm resources. The effects of different media, removal ages of immature seeds and reciprocal crosses of parents on the germination and seedling survival of immature seeds from crosses between diploid and tetraploid grape varieties by in vitro embryo rescue culture were investigated. The results indicated that the medium consisting of NN-1969 + IAA 1.75 mg l⁻¹ + GA₃ 0.35 mg l⁻¹ + CH 400 mg l⁻¹ + AC 2.0 g l⁻¹ was better than other media. The optimal removal age of immature seeds for the best development of embryos was 35–45 days after pollination (DAP). The percentage of germination (PG) for immature seeds and the percentage of seedling survival (PSS) for immature seeds for diploid varieties used as female parents were 10.72% and 4.35% higher than when tetraploid varieties were used as female parents respectively. A total of 41 hybrid progenies from eight combinations were obtained, made up of 17 diploid, 9 tetraploid, 14 aneuploid, and 1 triploid progeny as determined by root-tip chromosome identification. The triploid progeny was from Fujiminori (2n = 4x = 76) × Jingxiu (2n = 2x = 38). These results implied that it was feasible to extend the hybridization range of grape and to create new germplasm resources by in vitro embryo rescue based on the conventional hybridization. The NN-1969 medium supplemented with GA₃ and IAA was more propitious to the development of immature seeds sampled at about 45 DAP. It was easier to obtain plants using diploid as female parent, but triploid progeny was only obtained using tetraploid as female parent.     

In vitro propagation of <Emphasis Type="Italic">Epipactis flava</Emphasis> Seidenf., an endangered rheophytic orchid: a first study on factors affecting asymbiotic seed germination,seedling development and greenhouse acclimatization

Epipactis flava Seidenf., is an endangered Thai rheophytic orchid that has decreased rapidly throughout its natural habitat and urgently requires conservation using in vitro techniques. Effect of pollination types (self- and cross-pollination) and seed from different capsule ages (2, 4, 6 and 8 weeks after pollination) on asymbiotic seed germination efficiency was performed on semi-solid VW medium supplemented with 150 mL/L coconut water and 50 g/L potato extract. Highest germination rate was 79.2% with definite rate of protocorm developmental stage 5 at 24.0% using seeds from 6 week old cross-pollination capsules. Anatomical investigation revealed young seed has been formed at 6 weeks old capsules. Identical seeds were then cultured on semi-solid and liquid systems of five different media including modified-VW, MS, BM, MM and KC for 2 months. Results showed that semi-solid and liquid VW medium promoted the highest seed germination rates at 70.2% and 70.4%, respectively, with the highest definite rate of protocorm developmental stage 5 at 54% found on semi-solid BM medium. In vitro seedlings were transferred to culture on both semi-solid and liquid VW and MS media for 2 months. Higher growth parameters, as indicated by shoot number and fresh weight, were obtained on liquid MS medium. Clumps of plantlets containing 5–8 shoots were acclimatized and transplanted into different potting media for 3 months. Survival percentages in all tested substrates were recorded at nearly 100% with no significant differences, while 63–68% of the living plantlets produced new shoots.     

Rapid micropropagation via axillary bud proliferation of Adhatoda vasica Nees from nodal segments

A protocol for rapid multiplication of Adhatoda vasica has been developed through nodal explants from field grown mature plants. The maximum number of shoots, i.e., 7.75 +/- 0.392 differentiated from split nodal halves on MS medium supplemented with BA (10.0 mg/l) during 4 weeks of culture. Maximum number of shoots formed per explant increased to ca. 30 within 6 weeks of subculture on medium containing BA (1.0 mg/l) and Kn (1.0 mg/l). The isolated shoots rooted 90% in MS medium containing IBA (0.1 mg/l) in 2 weeks. The rooted plantlets were successfully transferred to soil in glasshouse and subsequently in field. The plantlets rooted in liquid medium did not survive, but those rooted on solid medium showed more than 75% survival. In vitro raised plants grew successfully ex vitro till flowering.     

温度和水分对不同基因型小麦未成熟胚体细胞胚胎发生以及分化能力的影响

对小麦未成熟胚盾片组织离体再生途径中,未成熟胚发育时期以及不同小麦品种的体细胞胚发生能力和体细胞胚的分化能力进行了研究,在所 试的14个小麦品种中,筛选出具有很强的体细胞胚发生能力和体细胞胚分化能力的4个品种,西农1376、盐2号、85＋1－3和宝丰7228。为进一步给小麦离体遗传操作打下基础,研究还对温度的影响进行了分析。通过低温手段解决了胚性愈伤组织随继代天数的延长体细胞胚分化能力快速降低的问题,同时研究还首次分析了干燥处理对小麦体细胞胚转换能力的影响,建立起一套高效的小麦离体培养再生体系,而且该体系从接种未成熟胚到再生植株移至土壤只需10－12周时间,避免了长期培养过程中存在的体细胞变异问题。     

Plant regeneration via somatic embryogenesis of Elymus sibiricus cv. ‘chuancao No. 2’

The mature seeds, mesocotyls, and young leaf tips of Elymus sibiricus L. cv. ‘chuancao No. 2’ were cultured on Murashige and Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.05 mg/L kinetin in the dark at 26°C, the calluses were produced. The rate of callus regeneration depended on the explants source and plant growth regulators. Plants regenerated from whitish-yellow-coloured compact nodular callus formed after subculturing for 8 weeks. Higher frequency (54%) of shoot differentiation was obtained from the embryo tissues of mature seed than from either mesocotyls (24%) or young leaf tip tissues (6%) when these calluses from different types of explants were cultured on plant regeneration medium containing half strength MS salts supplemented with 0.1 mg/L kinetin, 1.5 mg/L 2,4-D and 20 g/L sucrose. The green plants were rooted within 6 weeks in the root regeneration medium, and over 97% of these soil-established plants were obtained in the greenhouse when potted in a sand and peat mixture medium.