Small Plantlets Research Articles

Plantlet regeneration of Cucumis melo L. Glamour cv. using different types of cytokinin and explants

Plantlets or clones’ regeneration in Cucurbitaceae species can be performed in vitro by applying the tissue culture techniques. Hypocotyls, cotyledons, leaves, cotyledonary nodes and petioles can be used as the explants or starting materials to initiate the regeneration under laboratory condition. BAP (Benzyl aminopurine) and TDZ (Thidiazuron) were among the well-known cytokinin used in tissue culture as plant hormones to regulate the plant growth in vitro. The effectiveness of BAP and TDZ was investigated in this study to determine the effect of different types of cytokinin hormone on C. melo explants since both hormones might show differences in their activity towards C. melo explants. This study found that BAP was superior at all used concentrations compared to TDZ during most of the growing stage of the explants (cotyledons and hypocotyls). Based on this finding, the experiment was repeated by using different types of explants which were nodal explants, petioles and young shoots along with cotyledons and hypocotyls, by using BAP that showed excellent plantlet regeneration. Cotyledon and nodal explants successfully regenerated plantlets with excellent height and significant number of shoots compared to the rest of the explants. The plantlets from cotyledons and nodal explants were regenerated via direct regeneration without any formation of callus. The direct shoot regeneration was the preferred method for mass production via tissue culture because the chances of somaclonal variation is low. Contrarily, the hypocotyls, petioles and shoot tips mostly developed callus and only several treatments formed very small plantlets.

In vitro asymbiotic seed germination, protocorm formation, and plantlet development of Orchis simia Lam.: A threatened terrestrial orchid species

• Seed germination and plantlet growth were impacted by organic constituents and nitrogen sources. • Individual application of organic nitrogen compounds led to higher germination efficiencies. • The present protocol could be used for large-scale in vitro micropropagation of terrestrial orchids. The present study examined the seed morphometric and viability, asymbiotic seed germination, protocorm formation, and plantlet growth in Orchis simia Lam., an at-risk Euro-Mediterranean terrestrial orchid species. Furthermore, tissue histology pattern in vitro grown plantlets and field-established was compared. After one month, shoot and root primordia started to grow on developing protocorms and small plantlets became visible after 2–3 subculturing. Asymbiotic in vitro seed germination and plantlet growth attributes were significantly ( P <0.05) impacted by both type of main organic constituent and nitrogen sources. The seeds sown on media supplemented with pineapple juice (PJ) and casein hydrolysate (CH) had the highest germination percentage. In terms of required days to see the first signs of germination, CW+AV treatment brings about the fastest germination (6.8 ± 0.20 days). Protocorms grown on the media comprising PJ in combination with Aminoven (AV) or casein hydrolysate (CH) or Pep showed higher dimensions and therefore higher weight (% 450 increase) compared to protocorms grown on other media. Altogether, treatments with higher germination percentage, faster germination, and bigger protocorms finally resulted in better plantlet growth. Acclimatization of in vitro grown plantlets occurred by converting scattered vascular tubes into fully developed xylem and phloem vessels facilitating sap flow. Taking into account the fragmentation of O. simia populations in the Middle East and the Euro-Mediterranean basin mainly due to tuber overharvesting and climate change, the present procedure could be used for large-scale multiplication of in vitro raised seedlings to re-introduce into endangered regions.

Strawberry Living Mulch in an Organic Vineyard

A living mulch system can provide beneficial biodiversified phytocoenoses and spatial competition against weeds; however, it may also compete for water with the main cultivated crop under Mediterranean climate conditions. Strawberries employed as living mulch in a rain-fed hill vineyard of central Italy were evaluated for two years through a participative approach involving the farmer. A local wild strawberry was propagated by stolons to obtain small plantlets easily uprooted after the summer and then transplanted to a one-year-old vineyard. The densities of two and four strawberry plants per grapevine were compared with no living mulch in a randomized complete block design. A horizontal blade weeder was used once a year in all treatments. The results showed that strawberries as living mulch plus application of a blade weeder avoided the need for further soil tillage and assured a full soil cover during winter for both initial planting densities. The strawberry living mulch did not alter the grapevine transpiration during an incident of water stress in summer. Moreover, the yield per vine and the grape quality were comparable with those of the soil without living mulch. The growth of strawberry mulch was relevant in the area surrounding the vines. Furthermore, the living mulch guaranteed a constant soil cover reducing the risk for soil erosion while increasing the vineyard’s biological diversity. This may imply a higher resilience.

Advances in pineapple plant propagation

Abstract Pineapple is one of the most important fruits, with large production in tropical and subtropical regions and great appreciation by consumers all over the world. The pineapple plant has many specific morphological, anatomical and physiological characteristics that determine crucial aspects of pineapple crop management, such as flower induction, water use and vegetative methods of propagation. The use of sexual reproduction of pineapple is restricted to breeding purposes carried out by research institutes looking for new hybrids with improved agronomic characteristics. Seeds are only produced if cross pollination among varieties occurs. Commercially pineapple has to be propagated by vegetative material, an asexual reproduction, without new combinations of genes. Some types of propagules are naturally produced by the plants and called conventional planting material. Its availability and quality depend on many factors, especially cultivar and environment. Management techniques of this material have been continuously developed and will be addressed. In addition to the conventional planting material, which in many situations is not sufficient to assure expansion or at least maintenance of the cultivated area, several other methods of vegetative propagation of pineapple have been studied and made available along the last decades and will also be discussed, involving techniques of stem sectioning, apical growing point gouging and chemical treatment for transformation of flowers into plantlets. Stem sectioning has been especially interesting, as it is mostly done using plant residues available at low cost, and is a rather simple method suited for multiplication and production of disease-free planting material in nurseries. Gouging and chemical treatment are less practiced, but can be applied in ratoon crops, thereby avoiding the loss of the first cycle fruit. Chemical treatment usually results in rather small plantlets, that must be further grown in nurseries before planting them in the field. And finally micropropagation will also be focused, as in vitro production of plantlets is a very important method of multiplication of new pineapple varieties, but this method yet has not been transformed into a common commercial way of pineapple propagation due to the final high cost and to the still high risks of incidence of somaclonal variations among the plantlets produced.

IMPACT OF IMMATURE FRUIT SIZE AND GENOTYPE ON DIRECT CAULLOGENESIS IN MONOEMBRYONIC-SEED MANGO

The improved in vitro protocol of monoembryonic mango would facilitate the commercial micropropagation of cultivars and rootstocks. The nucellus tissue of mono-embryonic seeds of world-known mango cultivars ‘Saroli’, ‘Langra’ and ‘Chaunsa’ were used to induce the direct shoots. The simple procedure used for surface sterilization of the immature fruit resulted in 95% free-contaminants explants. The initial fruit size was found to be crucial for getting the DS within a short period of time (2 months). Full half of seeds from immature fruit without zygotic embryo were used as an initial explant. Browning of initial explants was controlled by anti-oxidants solution, activated charcoal containing-media and incubation in dark till maturation. The developed medium formula to induce cluster of shoots (0.5 cm long) was micro-salts of MS and macro of B5 supplemented with 4.52 I¼M 2,4-D and 9.83 I¼M 2iP within 3 months under dark. The appropriate fruit size used to induce the DS (caullogenesis) was 5-6 cm long. The smaller fruit size 1 cm long could not develop and died while the fruit size 2-4 cm long developed clusters of embryogenic callus under dark and 27±2°C. The induced clusters of shoots were cultured on the basal medium supplemented with 2.3 I¼M Kin and 0.49 I¼M 2iP in order to grow into small healthy plantlets under light and 27±2°C. Caullogenesis was detected only in ‘Langra’ and ‘Chaunsa’ whereas ‘Saroli’ explants failed throughout the two seasons’ experiment. The step-wise protocol and morphology of the induced DS were described in the current study.

Endophytic bacterium, Bacillus amyloliquefaciens, enhances ornamental hosta resistance to diseases and insect pests

A total of 84 bacterial endophytes were isolated from seeds of 6 cultivars of ornamental hostas, and they were identified to 5 species based on morphological characteristics and 16S rDNA sequence analysis. Among them, the strain ‘Blu-v2’, which was isolated from the seeds of cultivar ‘Blue Umbrella’ and identified to be Bacillus amyloliquefaciens, showed highest antifungal activity and capacity to deter feeding by Fall armyworms (Spodoptera fruigiperda). Lipopeptides in cultures of Blu-v2 were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and its antifungal activities were verified. However, the lipopeptide preparation did not show toxicity to larvae of Fall armyworms. In a greenhouse experiment, Blu-v2 was inoculated into small plantlets of hosta (cultivar ‘Rainforest Sunrise’). The leaves of plants with bacteria (endophyte-infected = E+) and without bacteria (endophyte-free = E−) were used in seven-day feeding experiments employing fourth-instar larvae of Fall armyworms. We found that there was a significant decrease in the weights of larvae fed with E+ compared to E− plants; and the mortality rate of larvae fed with E− leaves was lower (3.33%) compared to that of larvae fed with E+ leaves (30%). Based on our studies, we suggest that endophytic B. amyloliquefaciens strain Blu-v2 has potential value as a biocontrol agent to reduce damage from fungal diseases and insect pests of hosta cultivars.

Regeneración in vitro de brotes de Polianthes tuberosa L. a partir de yemas vegetativas de la inflorescencia y de tejido de cormo

Polianthes tuberosa L. es una planta endémica de México, comercialmente se cultiva para flor de corte, y, además, es utilizada en la industria farmacéutica y del perfume. Tradicionalmente, los productores la propagan utilizando los cormos, lo que ha ocasionado que el cultivo presente poca variabilidad genética y posiblemente, por esta razón solo se conocen cultivares con flores blancas. Con base en lo anterior el objetivo de la presente investigación consistió en establecer la metodología práctica y competitiva para propagar in vitro Polianthes tuberosa L. Las yemas y pequeños segmentos de tejido de cormo fueron colocadas sobre la superficie del medio de cultivo, base GC (comunicación personal Guillermo Carrillo) con las sales inorgánicas del medio de cultivo Murashige y Skoog (1962) por litro, 50 mL de agua de coco, 20 g de sacarosa, 6.4 g de agar y el pH fue ajustado a 5.7. Con este medio básico se prepararon medios de cultivo que contenían bencilaminopurina (BAP), ácido naftalenacético (ANA), ácido 2,4-diclorofenoxiacético (2,4-D), ácido indolacético (AIA), y cinetina. La formación de brotes que dieron origen a las plántulas ocurrió mediante, la regeneración directa de la yema floral la cual al crecer forma sólo una plántula y a partir del tejido de la base de la yema, de esta región se obtuvieron hasta seis brotes por yema en el medio que contiene BAP, 4.5 mg y ANA, 0.1 mg, del cual la regeneración de brotes fue mayor (56.1 %). En el caso de cormo el problema fue la contaminación llegando en algunos casos hasta el 100 % de los cultivos. Es de resaltar la importancia de poder lograr la regeneración masiva in vitro de plantas de nardo a partir de yemas florales.

Development of an in vitro protocol for a difficult-to-propagate endemic Australian dryland sedge species Mesomelaena pseudostygia (Cyperaceae)

In vitro propagation for Mesomelaena pseudostygia a difficult-to-propagate dryland sedge species (Cyperaceae) endemic to Western Australia is described. Multiple avenues to in vitro propagation were investigated: shoot culture, organogenesis and somatic embryogenesis, with zygotic embryos as initiation material. The highest multiplication rate for shoots was 3.4 ± 1.0 after 6 wk on basal medium (1/2 strength Murashige and Skoog) with 2.5 μM kinetin and 0.5 μM 6-benzylaminopurine. Shoots achieved peak rooting (83%) following a pulse treatment on basal medium containing 10 μM indolebutyric acid and 2 μM α-naphthaleneacetic acid for 7 wk, followed by transfer to medium (without growth regulators) for a further 7 wk. Alternatively, in vitro grown shoots were pulse treated on basal medium with both 100 μM indolebutyric acid and 20 μM α-naphthaleneacetic acid for 1 wk then placed in Rockwool plugs (under propagation house conditions) for another 7 wk resulting in 63% root induction. Rooted plantlets were also successfully transferred to potting mixture either in Rockwool plugs or bare rooted and maintained in propagation house conditions with ≥95% survival after 7 wk. These results indicate that micropropagation of M. pseudostygia is feasible for small to medium scale restoration purposes. The highest frequency of callus induction was from cultured zygotic embryos on basal medium with 5 μM α-naphthaleneacetic acid, whereas 2,4-dichlorophenoxacetic acid (2 or 5 μM) produced the largest callus sizes. A low frequency of shoot regeneration occurred in zygotic callus tissues in basal medium treatments containing cytokinin (kinetin or thidiazuron at 1 μM). A small proportion (<20%) of zygotic embryo callus explants from 2,4-dichlorophenoxyacetic acid treatments were found to be embryogenic, firstly developing embryo-like structures after 2 wk on basal medium (minus plant growth hormones), that continued to develop with approximately one in twenty germinating after a further 4 wk on basal medium to form small plantlets. Further optimisation is needed to improve somatic embryogenesis efficiency for mass propagation.

Regeneration of viable oil palm plants from protoplasts by optimizing media components, growth regulators and cultivation procedures

Oil palm protoplasts are suitable as a starting material for the production of oil palm plants with new traits using approaches such as somatic hybridization, but attempts to regenerate viable plants from protoplasts have failed thus far. Here we demonstrate, for the first time, the regeneration of viable plants from protoplasts isolated from cell suspension cultures. We achieved a protoplast yield of 1.14×106 per gram fresh weight with a viability of 82% by incubating the callus in a digestion solution comprising 2% cellulase, 1% pectinase, 0.5% cellulase onuzuka R10, 0.1% pectolyase Y23, 3% KCl, 0.5% CaCl2 and 3.6% mannitol. The regeneration of protoplasts into viable plants required media optimization, the inclusion of plant growth regulators and the correct culture technique. Microcalli derived from protoplasts were obtained by establishing agarose bead cultures using Y3A medium supplemented with 10μM naphthalene acetic acid, 2μM 2,4-dichlorophenoxyacetic acid, 2μM indole-3-butyric acid, 2μM gibberellic acid and 2μM 2-γ-dimethylallylaminopurine. Small plantlets were regenerated from microcalli by somatic embryogenesis after successive subculturing steps in medium with limiting amounts of growth regulators supplemented with 200mg/l ascorbic acid.

Pre-adaptation to Cu during Plant Tissue Culture Enhances Cu Tolerance and Accumulation in Begonia (Begonia evansiana Andr.)

A simple and efficient protocol was developed for culturing Cu-tolerant and Cu-accumulating plants via pre-adaptation to Cu during plant tissue culture. We induced multiple shoots from begonia (Begonia evansiana Andr.) leaf explants on MS medium supplemented with naphtaieneacetic acid and benzyladenine. After 3 months, small plantlets were transferred to MS medium supplemented with <TEX>$100{\mu}M\;CuCl_2$</TEX> for pre-adaptation to Cu and cultured for 5 months. Then, these plantlets were individually planted in pots containing artificial soil. An additional 500 mg of Cu dissolved in 1/4 strength MS solution was applied to each pot during irrigation over the course of 2 months. We planted pre-adapted and control begonias in soil from the II-Kwang Mine, an abandoned Cu mine in Pusan, Korea, to examine their ability to tolerate and accumulate Cu for phytore-mediation. Pre-adapted begonias accumulated <TEX>$1,200{\mu}g$</TEX> Cu/g dry root tissue over the course of 45 days. On the other hand, non-Cu-adapted controls accumulated only <TEX>$85{\mu}g$</TEX> Cu/g dry root tissue. To enhance Cu extraction, chelating agents, ethylenediamine tetraacetic acid (EDTA)-dipotassiun and pyridine-2,6-dicarboxylic acid (PDA), were applied. While the chelating agents did not enhance accumulation of Cu in the roots of control begonias, EDTA application increased the level of Cu in the roots of pre-adapted begonias twofold (to <TEX>$2,500{\mu}g$</TEX> Cu/g dry root tissue). Because pre-adapted begonias accumulated a large amount of Cu, mainly in their roots, they could be used for phytostabilization of Cu-contaminated soils. In addition, as a flowering plant, begonias can be used to create aesthetically pleasing remediation sites.

Somatic Embryo Formation on Mature Abies alba × Abies cephalonica Zygotic Embryo Explants

Somatic embryogenesis was achieved from mature embryos excised from stored hybrid Abies alba × Abies cephalonica seeds. Embryogenic tissue formation occurred on SH medium supplemented with 1 mg dm-3 benzyladenine. The formation of embryogenic tissue was influenced by the time of storage of seeds. Initiation frequencies 27.2 - 29.0 % were obtained in embryos isolated from 6 month and 1 year stored seeds. Embryos excised from 4-year stored seeds showed no response. Embryogenic structures appeared on the surface of hypocotyl. They originated without previous callus formation. Embryogenic tissues were maintained in long-term cultures. After maturation treatment cotyledonary somatic embryos developed and germinated in small plantlets.

Dry matter production of white clover (Trifolium repens L.), Caucasian clover (T. ambiguum M. Bieb.) and their associated hybrids when grown with a grass companion over 3 harvest years

Abstract The dry matter (DM) production of Trifolium repens, T. ambiguum and the backcross 1 (BC1) and backcross 2 (BC2) hybrids with T. repens as the recurrent parent were compared in mixtures with an intermediate heading variety of perennial ryegrass (Lolium perenne L.) under a cutting‐only management over 3 harvest years. Plots of parental legume species and backcross hybrids were established from small plantlets and oversown with the companion grass. In the first harvest year, the DM yield of clover in T. repens plots was greater than that in the BC2 plots and both greater than in the BC1 plots, whilst in the second and third harvest years differences between the DM yield of clover in the T. repens and the BC1 and BC2 hybrid plots were small. Similar results were obtained for the DM yield of total herbage. There were also differences in seasonal growth in the first harvest year, when yield of clover in T. repens plots was greater than in the BC1 and BC2 hybrid plots at early cuts but not at later cuts. Few differences in seasonal growth were observed between parental species and hybrids in subsequent harvest years. Comparison of above‐ and below‐ground biomass showed more DM in roots and rhizome of clover in the backcrosses than in the T. repens plots in the second harvest year but differences were less evident in the third harvest year. The clover in the backcross hybrid plots also had fewer stolon growing points per quadrat than the T. repens plots, but the BC2 had more than the BC1 plots. The exploitation of these hybrids in breeding programmes as a strategy to improve the persistence and drought tolerance of white clover is discussed and implications for forage production considered.

In vitro insect-feeding bioassay to determine the resistance of transgenic rice plants transformed with insect resistance genes against striped stem borer (Chilo suppressalis)

To determine the degree of insect resistance in transgenic plants, different bioassays are used which typically use either whole plant or small pieces of leaves or stems of transgenic plants, following culture under greenhouse conditions. An in vitro insect-feeding bioassay is presented which permits the infestation of transgenic plantlets with newly hatched larvae from the striped stem borer. The bioassay consists of the germination of rice seeds in vitro using Murashige and Skoog medium in test tubes, and then infestation of each 3–4 cm long seedling with one neonate larva obtained from surfacesterilized eggs of Chilo suppressalis. The infested in vitro plantlets are kept in culture rooms at 25°C for several days and then the seedling damage and the growth of the larvae are analyzed. Senia (japonica variety) homozygous transgenic rice plants were used for these experiments. The plants were transformed with either the cry1B or the maize proteinase inhibitor (mpi) genes. Both genes confer resistance to Chilo suppressalis. With non-transformed plants the larvae grew and developed normally, feeding on the small rice plantlets. In contrast, with cry1B plants, the neonate larvae died during the first days of the infestation. These plantlets recovered completely and developed similarly to the non-infested control plants. With transgenic plants transformed with the mpi gene, the neonate larvae did not die but grew more slowly compared with the controls. Thus, this in vitro insect-feeding bioassay is a rapid and easy method to detect the resistance of cry and mpi transgenic plants to stem borers such as Chilo suppressalis.

The morphological variability within a population of coffee somatic embryos produced in a bioreactor affects the regeneration and the development of plants in the nursery

A 1-liter bioreactor was used to obtain approximatively 800 Coffea arabica somatic embryos, 86% of which reached the `germinated' stage but with morphological heterogeneity. The population was sub-divided into three categories according to cotyledon area: 'small', 'medium' and 'large', that amounted to 32%, 36% and 4.5%, respectively. The effect of embryo morphology on plantlet conversion after direct sowing in soil and on plant development in the nursery was investigated. Somatic embryos with large cotyledons had only a 25% plantlet conversion rate, whereas somatic embryos with small to medium-sized cotyledons had conversion rates of 47% and 63%, respectively. The vigour of the aerial and root systems of regenerated plantlets at the end of the plant conversion stage was also affected as the embryos with small, medium and large cotyledon mostly regenerated small plantlets (0.5–1.5 cm), medium plantlets (1.5–2.5 cm) and large plantlets (2.5–5 cm), respectively. When transplanted in plastic bags, these 3 populations of plantlets exhibited distinct development rates. They had an initial slow growth phase, which was much longer for the small plantlets, followed by a rapid growth phase. After 40 weeks in the nursery, an analysis of the growth parameters of aerial and radical systems showed that the vigour of the plants was strongly related to the vigour of the plantlets transplanted. The heterogeneity of somatic embryos in the bioreactor affected both the plant conversion efficiency in soil and the plant growth in nursery, where it mainly resulted in retarded growth, primarily in plantlets derived from the somatic embryos with small cotyledons.

Rapid and efficient Agrobacterium-mediated transformation of Panax ginseng by plasmolyzing pre-treatment of cotyledons

A rapid and efficient genetic transformation of Panax ginseng cotyledon explants following a plasmolyzing pre-treatment was investigated. When cotyledon explants of P. ginseng were pre-treated with 1.0 M sucrose, transient expression of the β-glucuronidase (GUS) gene was strongly enhanced following co-cultivation with Agrobacteriumtumefaciens harboring the GUS gene. This enhanced expression coincided with a high frequency of stable transformation (three times higher than non-treatment). Blue-colored cells (indicative of the presence of the GUS gene) were detected over large areas of cotyledons pre-treated with sucrose. In contrast, when the plasmolyzing pre-treatment was not applied, GUS-positive cells were observed to be scattered on the cotyledons. Somatic embryos developed directly on cotyledon surfaces without intervening callus formation within 2 weeks and matured to the cotyledonary stage after about 7 weeks. Cotyledonary embryos regenerated into small transgenic plantlets on medium supplemented with gibberellic acid within 1 month. Thus, about 3 months of culture was required for small transgenic ginseng plantlets to be obtained. The presence of transformed ginseng plantlets was confirmed using the staining reaction of X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) and by the polymerase chain reaction.

Size of In Vitro Plantlets Affects Subsequent Tuber Production of Acclimated Calla Lily

Tuber production of calla lily (Zantedeschia elliottiana Spreng cv. Super Gold) was investigated using three size ranges (7-10, 4-7, and &lt;4 mm shoot diameter) of in vitro plantlets acclimated in either pots or soil beds in a protected house. The shoots and tubers of large plantlets exhibited higher rates of dry-matter accumulation than did those of small plantlets. The diameter of tubers harvested from pots ranged from 0.67 to 4.1 cm with median values of 2.7, 2.1, and 1.9 cm for the plants derived from large, medium, and small plantlets, respectively. Plants grown in soil beds, regardless of size, produced larger tubers than did those grown in pots. Tubers &gt;3 cm in diameter developed on 25% and 52% of plants grown in pots and soil beds, respectively. Our results suggest that improved calla lily production could be realized by using larger in vitro plantlets as the source material and growing them in soil beds in a protected house.

Date palm micropropagation in Saudi Arabia: policies and technology transfer

Saudi Arabia harbours one of the most important genetic centres of origin of date palm. Over the centuries this tree has been crucial for the survival of nomadic tribes in Saudi Arabia. Today, the fruits of this &quot;tree-of-life&quot; are highly valued as a local delicacy. Small plantlets purchased to replace non-productive palms and plantations infested with the red weevil, fetch high prices. This continuous demand has resulted in various Saudi public research initiatives to establish micropropagation protocols for the elite date palm varieties of Saudi Arabia. Together these institutes have succeeded in mastering the different elements necessary for the large-scale micropropagation of the date palm. Despite these efforts, it would take the input of a US company to transfer the technology for date palm micro-propagation in order to provide Saudi farmers with superior plantlets.

Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium tumefaciens-mediated transformation.

Embryogenic calli were induced from leaf explants of coffee (Coffea canephora) on McCown's woody plant medium (WPM) supplemented with 5 μM N6-(2-isopentenyl)-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase II genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentration (5, 50, 100 mg/l). The embryogenic calli surviving on medium containing 100 mg/l hygromycin showed a strong GUS-positive reaction with X-Gluc solution. Somatic embryos were formed from these putative transgenic calli and germinated on WPM medium with 5 μM 2-iP. Regenerated small plantlets with shoots and roots were transferred to medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the polymerase chain reaction.

Plantlet regeneration and bulbil formation in vitro from leaf and stem explants of Curculigo orchioides, an endangered medicinal plant

A method has been developed for rapid multiplication of Curculigo orchioides (Hypoxidaceae) through direct organogenesis and bulbil formation in vitro. Leaf and underground stem explants produced maximum number of shoots (four and 10 per explant, respectively) on B5 medium supplemented with 4.4 μM BAP. Higher concentrations of BAP (≥22 μM) in the medium completely inhibited the growth and shoot proliferation from the leaf explants. Stem-disc-derived callus produced numerous bulbils on the medium containing BAP (8.8–35.2 μM) and sucrose (0.12–0.23 M). Maximum number of bulbils (10 per explant, 0.19 g fresh weight per bulbil) were recorded on the medium supplemented with 35.2 μM BAP and 0.18 M sucrose. Isolated bulbils increased in size (maximum growth index 36.4) and produced secondary bulbils on transfer to medium of the same composition. A maximum number of secondary bulbils (six per bulbil) were produced on the medium containing 35.2 μM BAP and 0.23 M sucrose. Growth of in vitro produced bulbils into plantlets and rhizogenesis from isolated shoots was achieved on the medium devoid of plant growth regulators. Small plantlets developed in vitro were successfully (90% survival) transferred to the soil. Plantlets sprouted in the next season (June 1997) after over-wintering.

Seed-specific immunomodulation of abscisic acid activity induces a developmental switch.

A single-chain Fv antibody (scFv) gene, which has previously been used to immunomodulate abscisic acid (ABA) activity in transgenic tobacco to create a 'wilty' phenotype, was put under control of the seed-specific USP promoter from Vicia faba and used to transform tobacco. Transformants were phenotypically similar to wild-type plants apart from their seeds. Anti-ABA scFv embryo development differed markedly from wild-type embryo development. Seeds which accumulated similar levels of a scFv that binds to oxazolone, a hapten absent from plants, developed like wild-type embryos. Anti-ABA scFv embryos developed green cotyledons containing chloroplasts and accumulated photosynthetic pigments but produced less seed storage protein and oil bodies. Anti-ABA scFv seeds germinated precociously if removed from seed capsules during development but were incapable of germination after drying. Total ABA levels were higher than in wild-type seeds but calculated free ABA levels were near-zero until 21 days after pollination. We show for the first time seed-specific immunomodulation and the resulting switch from the seed maturation programme to a germination programme. We conclude that the immunomodulation of hormones can alter the development programme of target organs, allowing the study of the directly blocked endogenous molecules and manipulation of the system concerned.