Plant regeneration from embryogenic suspension cultures of Musa acuminata cv. Mas (AA)

Embryogenic callus was established using immature male flower of Musa acuminata cv. Mas. After 5–6 months of culture, embryogenic callus was obtained at 21.75±11.9 from 750 immature male flower clusters with translucent somatic embryos proliferated from the whitish friable callus. It was observed that flower clusters ranging from 4 to 11 responded to form embryogenic callus and out of which 3–10 somatic embryos were formed per flower cluster. Embryogenic callus were obtained at a percentage of 10.00±0.3 on M1 medium initially supplemented with 18 M 2,4-dichlorophenoxyacetic acid (2,4-D) for 3 months and subsequently transferred to the same media with reduced 2,4-D (9 M) for the next 2–3 months. Embryos developed into translucent spheres and slightly torpedo shaped embryos in suspension cultures. Plantlets were obtained on medium M4 supplemented with 0.8M BA, at an average regeneration rate of 13.00±0.58.     

影响体胚发生途径香蕉(Musa spp.,AAB Group)植株再生的因素

香蕉品种‘Agbagaba'和‘Orishele'的胚性细胞悬浮系(ECS)在液体培养基中分别预培养1和2周后,将其接种在RD1和M3培养基上,于光照或黑暗条件下进行体胚的再生.从沉积细胞体积(SCV)为1 mL(1 mL SCV)的ECS获得的再生体胚数量因预培养时间、再生培养基的种类及培养条件的不同而异.植株的再生率及从1 mL SCV的ECS获得的再生植株数量也受上述体胚再生条件的间接影响.     

Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration

A reproducible and efficient transformation method was developed for the banana cv. Rasthali (AAB) via Agrobacterium-mediated genetic transformation of suckers. Three-month-old banana suckers were used as explant and three Agrobacterium tumefaciens strains (EHA105, EHA101, and LBA4404) harboring the binary vector pCAMBIA1301 were used in the co-cultivation. The banana suckers were sonicated and vacuum infiltered with each of the three A. tumefaciens strains and co-cultivated in the medium containing different concentrations of acetosyringone for 3 days. The transformed shoots were selected in 30 mg/l hygromycin-containing selection medium and rooted in rooting medium containing 1 mg/l IBA and 30 mg/l hygromycin. The presence and integration of the hpt II and gus genes into the banana genome were confirmed by GUS histochemical assay, polymerase chain reaction, and southern hybridization. Among the different combinations tested, high transformation efficiency (39.4 ± 0.5% GUS positive shoots) was obtained when suckers were sonicated and vacuum infiltered for 6 min with A. tumefaciens EHA105 in presence of 50 μM acetosyringone followed by co-cultivation in 50 μM acetosyringone-containing medium for 3 days. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into banana has been developed and that this transformation system could be useful for future studies on transferring economically important genes into banana.     

贡蕉胚性细胞悬浮系的建立和植株再生

鲜食蕉品种的高度不育性和多倍性制约了用传统育种方法培育生产实践中所需的新品种 ,建立稳定的胚性细胞悬浮系是香蕉生物技术育种的前提。以目前国内尚未建立该体系的鲜食蕉品种贡蕉 (AA)未成熟雄花序的第 1～ 15位花梳为外植体 ,对胚性细胞悬浮系的建立和植株再生体系进行了优化。结果表明 ,5～ 6个月的培养后可获得分生小球体和浅黄色、松散易碎的胚性愈伤组织。 9μmol/L 2,4 D对外植体愈伤组织的诱导效果最好 ,诱导率为 40.96 % ,胚性愈伤组织诱导率可达7.45 % ,其中5.79%的胚性愈伤组织来源于第 6～12号位置的花梳。胚性愈伤组织悬浮培养后 ,通过 3个月的筛选和继代培养 ,可得到均质的胚性细胞悬浮系。该培养体系合适继代周期为 15d ,继代时合适的起始接种量为每 30mL培养基加 2mLPCVECS。培养 6个月的胚性细胞在体细胞胚诱导培养基中培养15d后可见到白色半透明体细胞胚的发生 ,体细胞胚诱导率为 2 80× 10³个 mLPCV。成熟体细胞胚的萌发率为 17 2 8% ,其中发育成正常的再生植株的百分率为 14 16 %。     

Desiccated quiescent somatic embryos of orchardgrass for use assynthetic seeds

Summary Somatic embryos of orchardgrass became quiesent when desiccated to 13% water. Twelve percent germinated after 21 d of desiccated storage at 23° C and 4% developed into green plants. During desiccation, embryos decreased in size, became yellowish and brittle, and their outer walls collapsed. Within 15 min after imbibition, they rapidly enlarged and were indistinguishalbe from nondesiccated embryos. These results suggest that somatic embryos may be engineered to function as synthetic seeds for mass propagation. This research represents a collaborative effort between the Agricultural Experiment Stations of the University of Florida and the University of Tennessee. Research was supported in part by the Competitive Research Grants Office of the U.S. Department of Agriculture, grant 82-CRCR-1-1086, awarded to the University of Tennessee. Florida Agricultural Experiment Station Journal Series No. 7173.     

Amplified somatic embryogenesis from male flowers of triploid banana and plantain cultivars (Musa spp.)

Summary Somatic embryogenesis and plant regeneration of banana and plantain cultivars (Musa spp.) were obtained by culturing young male flowers. Multiplication and maintenance of embryogenic cultures were achieved by culturing somatic embryos in a temporary immersion system (SIT). A multiplication rate of 40 allowed us to obtain more than 6000 somatic embryos after 6 mo. of subculture. Plant recovery frequencies were 60 to 70%. This method was expanded to different banana and plantain genomic groups.     

Histology of somatic embryo initiation and organogenesis from rhizome explants of Musa spp.

The origin of somatic embryos derived from rhizome explants of triploid Musa cv. Grand Nain was the subject of histological studies during different phases of ontogenetic development. The investigation revealed that the majority of somatic embryos showed normal root formation and consisted of highly vacuolated cells in the poorly structured shoot apex. The embryogenic mass and somatic embryos were mostly derived from several morphologically competent cells. Single cell origin depended on the presence of organogenetically functional vascular cells of rhizome explants and occurred infrequently. The implications of these findings for genetic improvement of banana and plantain by in vitro mutation breeding and gene technology are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction and development of somatic embryos from spinach (Spinacia oleracea) leaf segments

Somatic embryogenesis was induced in embryogenic calli of Spinacia oleracea L., on a Murashige and Skoog (1962) medium, containing 4.6 μM kinetin as the sole growth regulator. Abscisic acid, gibberellic acid, and indole-3-acetic acid were supplemented to kinetin-containing medium and their effects on the initiation of somatic embryos was studied. Abscisic acid at a particular concentration (4 μM) dramatically increased the number of embryos per g fresh weight of callus, while both gibberellic acid and indole-3-acetic acid suppressed the embryo initiation. It is suggested that the promoting effect of abscisic acid on the embryo initiation may be explained as a stress response of the tissue. The relative number of globular embryos vs. the embryos in heart/torpedo and cotyledonary stages was increased at 4 μM abscisic acid and at all gibberellic acid concentrations (0.3–10 μM). In contrast, the ratio of globular to polar embryos was lower than in controls at 1 μM abscisic acid and indole-3-acetic acid (1 and 10 μM). The effects of growth regulators on the ratio of globular to polar embryos indicate that they interfere with the normal distribution of cell division and cell expansion during early embryogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Low temperature storage of suspension culture-derived grapevine somatic embryos and regeneration of plants

Summary A method of clonal germplams preservation utilizing dehydrated somatic embryos and cool temperature storage conditions was demonstrated. Somatic embryos of grapevine (Vitis vinifera L) Autumn Seedless and Chardonnay were produced from suspension cultures. After washing twice with sterile water mature somatic embryos were blot-dried and placed on sterile filter paper in an open Petri dish in a laminar flow hood until they reached about 25% of their initial weight. Approximately 300 dried embryos were placed in each sterile 90×15 mm Petri dish, which was tightly sealed with two layers of Parafilm^TM. Sealed dishes were stored in the dark at 4°C in a standard refrigerator. Samples of 25–60 individual dehydrated somatic embryos were periodically tested for viability by placing them on solidified MS medium for germination and plant regeneration. After 42 mo. of dehydrated storage, 90% of the somatic embryos regenerated into plants. To further test utility, of this storage method dehydrated embryos stored for 12 and 26 mo. were shipped from Florida to Washington where 75 and 87.5% regenerated into plants, respectively. Cool temperature storage of dehydrated somatic embryos is a simple and inexpensive method of clonal, germplasm preservation when compared to alternatives such as cryopreservation.     

Growth Parameters of Yield of Plantain (Musa cv. AAB)

The performance of the crop plant and the ‘ratoon’plant (lateral shoot succeeding the harvested main shoot) of65 medium-sized False Horn plantains (Musa cv. AAB) was studiedby measuring vegetative and inflorescence (bunch) parameters.From significant regressions between different parameters itappears that taller pseudostems produce leaves at a faster rate,flower earlier and produce heavier infructescences which needmore time to mature. A high yield is determined by vigorousinitial growth of the planted sucker (lateral root). Beforeflowering, there is no sink competition between ratoon and mainpseudostem growth, and fast-growing main pseudostems are accompaniedby fast-growing ratoons. After flowering, competition occursbetween the fructifying inflorescence, a preferential sink andthe ratoon. The results indicate that plantain should not beconsidered as an annual crop. Growth, development, production parameters, bananas, plantains     

Production of isolated somatic embryos from sunflower thin cell layers

We describe here a two step procedure which allows the easy isolation of somatic embryos from Sunflower (Helianthus annuus L.) hypocotyl tissues. Thin cell layers composed of the epidermis plus 3 to 6 parenchyma cell layers were incubated for 5 days in a basal Murashige and Skoog medium using an auxin to cytokinin weight ratio of 1/1. The epidermis layers were then transferred to a Gamborg medium containing a high level of sucrose. After one week of incubation in this medium, many somatic embryos started to be released from the parental epidermal tissue. Even though the germination of these embryos is difficult, we have been able to induce secondary embryos and regenerate fertile plants.Abbreviations NAA 1-naphthalene acetic acid - IAA indole-3-acetic acid - BAP 6-benzylamino-purine - MS Murashige and Skoog medium - B5 Gamborg medium     

Plantlet regeneration from encapsulated somatic embryos of hybrid Solanum melongena L

High frequency somatic embryogenesis was induced from leaf expiants of F1 hybrid Solanum melongena L. on Murashige and Skoog's medium supplemented with 8.0 mg/1 NAA and 0.1 mg/1 Kn. The somatic embryos were encapsulated in various concentrations (2–6%) of sodium alginate and complexed with calcium chloride (25–100mM): 3% sodium alginate and 75 mM calcium chloride were found to be optimal for encapsulation. The encapsulated somatic embryos were transferred to various conversion media in vitro and in vivo. The frequency of plantlet regeneration varied from 27.0–49.7% in vitro and 2.0–4.5% in vivo.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid     

Production of cocoa plants (Theobroma cacao L.) via micrografting of somatic embryos

Summary The possibilities for in vitro regeneration of cocoa plants have been limited. Somatic embryos can differentiate from cotyledonary cells, but their conversion into plants has remained largely unsolved. In the present study, we attempted micrografting of somatic embryos to seedling rootstocks. Different conditions were analyzed for the rootstocks and embryos, and it was found that complete plant regeneration eeeded about 10 mo. The best results were obtained using a simple culture medium, 3-wk-old rootstocks, and somatic embryos without cotyledons. Histologic events associated with the graft union were also analyzed. Cells, mainly from the rootstock, initiate cellular division in different patterns, producing a callus at the graft junction. Within this region some cells differentiated into zylem and phloem, establishing vascular connection in the graft. Afterward, micrografted plants start to grow and differentiate new leaves and roots allowing for transfer to soil.     

Regeneration of somatic embryos from sweet orange (C. sinensis) protoplasts using semi-permeable membranes

Sweet orange (C. sinensis L. Osbeck) protoplasts were isolated from nucellar-derived embryogenic callus, cultured in alginate beads for 5–30 days, and the resulting p-calli released by liquefaction and cultured on semi-permeable membranes overlaid on MT culture medium. Somatic embryos did not develop from 5- to 10-day-old p-calli but did develop from 15-, 20-, 25-, and 30-day-old p-calli. There were no significant differences in the numbers of embryos produced among the 15- to 30-day-old p-calli and no abnormal embryo morphologies were observed. The minimum size of p-calli to form embryos was 77.84 μm in diameter. Embryos were smaller from p-calli than those produced from embryogenic callus; p-calli-derived embryos ranged in size between 0.5 and 0.8 mm, while embryos derived from embryogenic callus ranged between 1 and 2 mm.     

Plant regeneration from cultured protoplasts of the cooking banana cv. Bluggoe (Musa spp., ABB group)

Summary Suspensions of embryogenic cells of a triploid banana (Musa spp., cv. Bluggoe) were initiated from the uppermost part of meristematic buds, and used as protoplast source. After 20 weeks in culture, the suspension contained a mixture of globular structures or globules and embryogenic cell clusters, as well as single cells. Two types of protoplasts were obtained from embryogenic suspension culture: small (20–30 m) and larger (30–50 m) protoplasts with a dense cytoplasm and large starch grains respectively. The small protoplasts probably originated from embryogenic cell clusters, and also from pseudocambial cells of globules, while larger protoplasts were probably released from oval starchy cells and those of the globule peripheral area. In co-culture with a suitable feeder, consisting of suspensions of diploid banana cells, the protoplasts of triploid banana reformed the cell wall within 24 h and underwent sustained divisions leading to the formation of small clusters of 2–3 cells within 7 days. The latter developed directly into embryos without passing through an apparent callus phase. 10% of such embryos gave rise to plantlets when subcultured in 2.2 M 6-benzylaminopurine and 2 M 4 amino-3,5,6-trichloropicolinic acid for 1 week, before transfer to MS medium containing 10 M 6-benzylaminopurine. The rest of the embryos underwent intensive direct secondary embryogenesis which could lead to the formation of plantlets with a frequency of up to 50% upon further transfer to hormone-free medium.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog (1962) medium - 2,4-D dichlorophenoxyacetic acid - UV ultraviolet light - FDA fluorescein diacetate - MES 2-(N-morpholino)ethanesulfonic acid - Picloram 4 amino-3,5,6-trichloropicolinic acid     

Direct seeding of grapevine somatic embryos and regeneration of plants

Summary Somatic embryos of grapevine (Vitis vinifera L.) ‘Chardonnay’ were produced from liquid suspension cultures. Mature somatic embryos were blot dried briefly in the laminar flow hood and germinated directly in Magenta GA-7 Vessels^™ containing one of the following potting media: (1) sand, (2) commercial potting mixture (CPM), or (3) CPM overlaid with sand. Each vessel containing 20 ml of distilled water and the potting medium was sterilized by autoclaving for 30 min and cooled overnight before inoculating the somatic embryos. Five somatic embryos were placed in each vessel under aseptic conditions. The vessels were closed and incubated at 26±2°C, 16 h photoperiod at 75 μmol s⁻¹ m⁻² light intensity. Results revealed that CPM overlaid with sand was best for plant development. There was more contamination of somatic embryos on pure CPM. Since direct seeding bypasses at least two subcultures in agar medium, it has implications for use of somatic embryos as ‘synthetic seeds’ for clonal plant production. This study shows that somatic embryos of grapevine can be handled with some of the convenience of seeds, emphasizing the feasibility for further automating in vitro plant production, which might be especially useful for new varieties where propagation material is limited.     

Plant recovery from maize somatic embryos subjected to controlled relative humidity dehydration

Summary Maize (Zea mays L.) embryogenic type-II calli were grown on medium containing 0,0.1 μM ABA or 60 g/liter sucrose or both before dehydration of solitary somatic embryos under three relative humidity regimes for up to 6 wk. Viability of dehydrated embryos after 2 wk rehydration was assessed by their ability to produce chlorophyll (greening), roots, coleoptiles, and/or leaves. Only embryos sequentially pretreated with ABA and high sucrose remained viable after 2 wk of dehydration at 70% RH. Up to 34% of the somatic embryos survived 2 wk dehydration at 70% RH, whereas embryos dehydrated at 50 or 90% RH exhibited reduced viability (8.7 and 0.8%, respectively). Approximately 15% of the embryos dehydrated at 70% RH developed into plants, whereas 0.9 and 0% of embryos dehydrated at 50 and 90% RH produced plants. Three percent of maize somatic embryos remained viable after 6 wk of dehydration at 70% RH, and 1.7% developed into plants. Embryo size influenced the ability of maize somatic embryos to survive dehydration. Only embryos greater than 5 mm survived 2 wk dehydration at 70% RH.