Few studies on embryogenesis in common bean (Phaseolus vulgaris L.) have been reported and only the early stages of somatic embryogenesis were observed. Dry seeds from two common bean lines were germinated in darkness on L-6 medium containing 4% sucrose, 0.2 g casein hydrolysate /liter and 2.0 g phytagel /liter. The medium for seed germination was supplemented with 0, 2, 4 or 6μM forchlorfenuron (CPPU). Explants from cotyledonary leaves, petioles, hypocotyls and shoot apices were prepared from 14 day-old seedlings. Callus was derived from explant cultures incubated in darkness at 26C on the medium containing 4 μM 2,4-D and 1 μM Kinetin. The callus was transferred after 4 weeks into 125 ml Erlenmeyer flasks containing 50 ml liquid medium and placed on a gyrotary shaker (120 rpm) under cool-white light (12 μmol.m-2.s-1). The liquid medium was used with 2, 4 or 6 μM of 2,4-D alone or with zeatin supplements at relative concentrations of 0.25 and 0.5. Up to 200 somatic embryos from 40 to 50 mg callus inoculations were induced after 4 to 5 weeks. Callus derived from seedlings grown on CPPU-containing medium gave more repetitive somatic embryos. Cotyledonary stage embryos with clear bipolar structure were observed only from callus derived from seedlings grown on CPPU when transferred to suspension cultures containing 2,4-D and zeatin. All somatic embryos differentiated strong roots and some developed leaf-like structures on conversion medium.
Mohamed F. Mohamed, Paul E. Read, and Dermot P. Coyne
Abdoulaye Traore and Mark J. Guiltinan
The effects of five carbon sources (glucose, fructose, maltose, sorbitol, and sucrose) and two explant types (petals and staminodes) on cacao somatic embryogenesis was studied. No growth was observed on both types of explants cultured on sorbitol containing media and slow growth was obtained on media supplemented with maltose. Depending on the genotype, the percentage of explants producing one or more embryos ranged from 6% to 99%, 18% to 98%, and 3% to 82% on media containing glucose, fructose and sucrose respectively. Explants cultured continuously on maltose or sorbitol-containing media failed to produce embryos. Staminode explants produced 3 to 10 times more somatic embryos than petals. A strong genotypic effect on somatic embryogenesis was observed. Staminode explants of the Forastero clones Laranja and PSUSca 6 produced 2 to 30 times more somatic embryos than the Trinitarios UF 613 and ICS 16. During embryo maturation and conversion, no significant differences were observed among glucose, fructose, maltose, or sucrose for embryo weight, total shoot and root production. However, we found that all plantlets produced on glucose had shoots with normal cacao leaves while the other carbon sources sometimes produced plantlets with cotyledon-like leaves.
Qian Zhang, Jianjun Chen, and Richard J. Henny
Homalomena `Emerald Gem' is an important ornamental foliage plant and widely used for interior plantscaping. Current propagation of this cultivar has been primarily carried out through in vitro culture by organogenesis; regeneration through somatic embryogenesis has not been documented. This report describes successful plant regeneration via direct somatic embryogenesis from explants of different organs. Somatic embryos formed at and around the cut surface of petiole, spathe, and peduncle explants. Embryos also appeared at the base between expanded ovaries of the spadix segment, and around midrib of leaf explants. The optimal treatments for somatic embryo occurrence from petiole, spathe, and peduncle explants were MS medium containing 0.2 mg/L NAA or 0.5 mg/L 2, 4-D with 2.0 mg/L CPPU, and for spadix explants were MS medium with 0.5 mg/L PAA and 2.5 mg/L TDZ. Somatic embryos appeared 6 to 8 weeks after culture and formed large embryo clumps in 3 to 4 months. Somatic embryos produced more secondary embryos and geminated on induction medium. Multiple shoot development and plant regeneration occurred from somatic embryo clusters on MS medium without hormone or with 2 mg/L BA and 0.2 mg/L NAA. The regenerated plants grew vigorously after transplanting to a soilless container substrate in a shaded greenhouse.
Qian Zhang, Jianjun Chen*, and Richard Henny
Pothos (Epipremnum aureum Linden & Andre), a climbing vine with leathery, shiny-surfaced, solid green or variegated heart-shaped leaves, is widely grown as an ornamental tropical foliage plant in hanging baskets or on poles as climbers for interiorscaping. Since pothos easily develops roots from nodes, its propagation is mainly from eye cuttings. Eye cuttings, however, frequently carry diseases from stock plants into production greenhouses. The objectives of this study were to investigate if somatic embryogenesis could be induced from a common cultivar `Golden Pothos' and germinated somatic embryos could be a means of clean propagule production. Using a modified MS medium supplemented with 2 mg·L-1 CPPU or TDZ and 0.2 mg·L-1 NAA or 0.5 mg·L-1 2,4-D, somatic embryos formed directly at cut edges of leaf explants, around petiole and stem explant ends, and along their side surfaces. Most somatic embryos maturated and grew into multiple buds or shoots; some of them developed into whole plants on the original medium. Somatic embryos also germinated and developed into plants on MS medium containing 2 mg·L-1 Zeatin and 0.2 mg·L-1 NAA, MS or 1/2 MS containing 2 mg·L-1 BA with or without 0.2 mg·L-1 NAA. Shoots elongated and roots grew on PGR-free medium. Plantlets grew healthy in shaded greenhouses after transferring to soilless substrates. This study suggests that the established method of somatic embryogenesis can be used to generate disease-free propagules of pothos for production.
Frederick G. Gmitter Jr. and Xubai Ling
A method was developed to produce nonchimeric, autotetraploid Citrus plants via in vitro somatic embryogenesis in the presence of colchicine. Undeveloped ovules from immature fruit of `Valencia' sweet orange (Citrus sinensis [L.] Osb.) and `Orlando' and `Minneola' tangelos (Citrus reticulata Blanco × Citrus × paradisi Macf.) were held on Murashige and Tucker medium with 500 mg malt extract/liter and 0.0090, 0.01%, or 0.10% colchicine for 21 days. Embryogenesis from tangelo ovules was suppressed by 0.10% colchicine, but no such effect was observed among sweet orange ovules. Colchicine treatments had no subsequent effect on embryo germination. The numbers of chromosomes in root tip cells showed that both tetraploid and diploid `Valencia' and `Orlando' plants were recovered from colchicine treatments. `Minneola' cultures produced only diploid plants. Tetraploid plant morphology was typical for Citrus tetraploids. Examination of chromosome numbers in root tip, shoot, and leaf meristems indicated that the regenerants were nonchimeric. Such nonchimeric tetraploids will be useful parents for interploid hybridization directed toward development of seedless triploid Citrus scion cultivars.
Paula P. Chee
A simple procedure for regeneration of cucumber plants (Cucumis sativus L. cv. Poinsett 76) from cotyledon and hypocotyl explants has been developed. Somatic embryogenesis was induced on Murashige and Skoog (MS) salts and vitamins medium supplemented with 2,4-D at 2.0 mg·liter-1 and kinetin at 0.5 mg·liter-1. Development of embryos was accomplished on MS medium with NAA at 1.0 mg·liter-1 and kinetin at 0.5 mg·liter-1. Eighty-five percent of the mature somatic embryos formed showed a typical bipolar structure. All developed into morphologically normal plantlets when transferred to MS medium containing no growth regulators. Chemical name used: 2,4-dichlorophenoxyacetic acid (2,4-D).
Hyo-Geur. Park, Kyung-Young Choi, and Do-Hyun Lee
Hot pepper is the most important vegetable in Korea in terms of both acreage and product value. Morphogenic response in tissue culture of hot pepper varied greatly with explant source and growth regulators. Somatic enbryogenesis, which we believe had not been reported yet, was only induced from 5-month-old callus derived from anther culture. Embryogenic callus was solely observed in the MS medium supplemented with 2 mg/l of NAA and 2 mg/l of BA. Better somatic embryogenesis and plant regeneration was observed on hormone free medium. Adventitious organogenesis was well induced from both hypocotyl and cotyledon. Polarity of explant for shoot regeneration was observed. Success rate of regeneration has been continuously improved mainly due to combinations of growth regulators. The best combination so far was 2 mg/l of zeatin and 1 mg/l of IAA, resulting in 92% of regeneration from cotyledon explant.
C.K. Kim, J.Y. Oh, J.D. Chung, A.M. Burrell, and D.H. Byrne
Somatic embryogenesis was initiated from in vitro-grown leaf explants of rose using an induction period of 4 weeks on MS basal medium supplemented with auxin followed by several subcultures on MS basal medium with cytokinin. `4th of July' showed the highest regeneration frequency (24.4%) on 5.3 μm NAA followed by culture on medium containing 18.2 μm zeatin. `Tournament of Roses' produced somatic embryos when cultured for 4 weeks on medium containing dicamba, 2.3 μm followed by three subcultures on medium containing 18.2 μm zeatin. Embryogenic callus matured on MS media containing 0.5 μm NAA, 6.8 μm zeatin, and 2.9 μm GA3. Long-term cultures were established for both cultivars. Somatic embryos germinated on MS medium containing IBA and BA. Silver nitrate (58.8 μm) enhanced shoot formation and germination of somatic embryos. Plants derived from somatic embryos were acclimatized and successfully established in the greenhouse.
Jae-Dong Chung, Hong-Yul Kim, Jung-Hae Suh, Oh-Chang Kwon, and Chang-kil Kim
Somatic embryo formation was observed on thin-sectioned leaf explants within 3 weeks of culture from two Phalaenopsis hybrids—Phalaenopsis Hwafeng Redjewell `Ching Ruey' Phalaenopsis Chingruey's Giant Ching Ruey' (R×R), and Phalaenopsis Formosa Best Girl Ching Ruey' Depts. Lih Jiang Beauty `S 566' (WR×WR). Frequency of somatic embryo formation was higher in hybrid WRxWR than R×R and optimal concentration of TDZ for the induction of somatic embryos was 9.08 μM. In (WR×WR) embryo proliferation was simultaneously observed after transferring the explants with somatic embryo clumps onto PGR-free half-strength MS medium. Six months after initiation, the culture plantlets were produced. This is the first report on somatic embryogenesis induced directly from the leaf explants using TDZ in Phalaenopsis.
Lianghong Chen, Ajmer S. Bhagsari, Soon O. Park, and Sarwan Dhir
This study was carried out to optimize conditions for plant regeneration of sweetpotato [Ipomoea batatas (L.) Lam] using shoot tips, petioles, and leaves of Selection 75-96-1 as explants in Murashige and Skoog (MS) with several growth regulators at different levels. Callus initiation and callus proliferation media were 9.0 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 9.0 μm 2,4-D + 1.1 μm N 6-benzyladenine (6-BA) in protocol I; 8.1 μm α-naphthaleneacetic acid (NAA) + 1.2 μm kinetin (KIN) and 5.4 μm NAA + 4.6 μm KIN in protocol II; 0.9 μm 2,4-D, and 0.9 μm 2,4-D + 1.2 μm N-isopenylamino purine (2iP) in protocol III; NAA (8.1 μm) + KIN (1.2 μm) and 2,4-D (0.9 μm) + 2ip (1.2 μm) in protocol IV, respectively. In protocol I and II, shoot tip, petiole, and leaf were used, but only petiole and leaf in protocol III and IV. In the protocol I and II, somatic embryos were obtained only from shoot tip explants; in protocol III and IV, only from petioles. The frequencies of somatic embryo development were 33.3% in protocol I, 42.1% in protocol II, 21.2% in protocol III, and 10.3% in protocol IV, respectively. The leaf explants failed to produce somatic embryos in all the experiments. In protocol I, somatic embryogenesis occurred through the well-known sequence of globular-, heart-shaped-, torpedo-, and cotyledon-type embryos. However, in protocol II, the structures resembling plumule and radicle were observed before the emergence of torpedo/cotyledon type embryo clusters. The somatic embryogenesis in protocol III and IV was similar to that in protocol I. Growth regulators influenced somatic embryo development. Further, this study showed that explant resource and growth regulators affected the frequency of plant regeneration in sweetpotato.