Production Of Artificial Seeds Research Articles

SYNTHETIC SEED TECHNOLOGY, APPLICATION AND FUTURE TRENDS

In nature, seeds are typically the primary method of plant propagation. Some plants can be vegetatively propagated but conventional methods are time consuming, expensive and cannot produce plants at larger scale. Synthetic seed technology could play a significant role in the production of artificial seeds, or Syn-seeds. Somatic embryos lacked crucial auxiliary tissues like endosperm and protective coverings, which made them difficult to handle and store. Two kinds of somatic embryos are known as desiccated and hydrated seeds based on the techniques developed thus far. The goal of this study is to emphasize the historical and current status of the creation of synthetic seeds. The micropropagation method is still one of the main barriers to the advancement of artificial seed technology. Despite the use of somatic embryos for artificial seed generation in a variety of plant species, there are still some significant problems that must be resolved before progress can be made. The synthetic seed process is a godsend for the vegetative multiplication, conservation, & long-term conservation of rare, endangered, and vulnerable species' superior germplasm. The current scenario of artificial seed to advance agriculture innovation still requires more practical directed appliances.

Application of temporary immersion system RITA® for efficient biomass multiplication and production of artificial seeds for ex situ conservation of Linnaea borealis L

Linnaea borealis L., belonging to the Linnaeaceae family, in some European countries including Poland, is a protected species. Phytochemical studies indicate the presence of pharmacologically important secondary metabolites, which may indicate undiscovered biological activity and untapped therapeutic potential of this species. Harvesting the raw material from natural sites is impossible due to the protection status of this glacial relict in many countries. The aim of the current work was to develop an efficient protocol of Linnaea borealis L. var. borealis shoot multiplication via the development of axillary buds in an agitated culture and a temporary-immersion system as well as to establish the production of artificial seeds via alginate encapsulated nodes for short-time storage. The highest results of growth parameters were obtained for shoots agitated in MS (Murashige & Skoog) liquid medium enriched with cytokinin, auxin and gibberellin (18.3 ± 0.4 shoots per explant and 2185 ± 98% biomass increase, respectively). The growth of shoots in the RITA® bioreactor was highly efficient, especially when hormone supplementation in MS medium was used, the amount of medium in the culture vessel was 100 or 150 ml, and the immersion time of the shoots was 1 or 2 min. The survival and recovery rates of L. borealis artificial seeds, regardless of encapsulation matrix used, subsequently decreased with the increased storage duration up to 6 months (from 100 to 60% at 4 °C and from 100 to 54% at -18 ºC, respectively). This is the first report on twinflower shoot multiplication in a temporary-immersion system and artificial seeds production of this species.

A comparative study on the effects of ultrasound and some growth factors on somatic embryogenesis and artificial seed production in cucumber (Cucumis sativus L.)

Cucumber (Cucumis sativus L.), a popular vegetable crop of the family Cucurbitaceae, is cultivated in different parts of the world and is of great economic importance. Ultrasound is known as a physical stimulus that can affect living systems. At the right frequency and exposure period, these waves have desired effects. In the present study, 2-week-old plants, were put in an ultrasonic bath with a nominal frequency of 40 kHz, a central frequency of 34/722 kHz and a bandwidth of 320 Hz for 0, 5, 10 and 15 minutes. Hypocotyl pieces were used as explants and cultured in Murashige and Skoog medium supplemented with 0/5 mg/l each of 2,4-dichlorophenoxyacetic acid, 1-Naphthalene acetic acid, kinetin and 6-benzylaminopurine. The use of ultrasonication at a nominal frequency of 40 kHz for 10 minutes showed better results as compared with the samples treated for 0, 5 and 15 minutes, in terms of the shape, maturity of the embryos, and also the germination of artificial seeds and maturing to flowering stage. Since the cucumber of Esfahan is very famous for its aroma and taste and it is somewhat at risk of extinction, we studied the production of artificial seeds of this valuable plant using somatic embryos and apical buds. This research is the first report on investigation of the positive effect of ultrasound on somatic embryogenesis and artificial seed production. our results clearly showed that this method is a practical method to accelerate seed germination and flowering stage.

Study on Artificial Seeds of Plants

The establishment and development of artificial seed technology is to quickly reproduce excellent varieties or hybrids, which can be applied to hybrid generation seeds produced by the three-two line method. For some varieties that are difficult to propagate with seeds or plant species with unstable genetic traits and poor fertility, artificial seed technology can also be used for mass reproduction. In particular, some new plants created through genetic engineering, such as somatic hybrids or transgenic plants, can be propagated or maintained by artificial seed technology. In addition, artificial seed technology can be used for the maintenance and rapid propagation of virus-free seedlings. Compared with ordinary test tube seedlings, artificial seeds have low cost, convenient transportation, and to a certain extent reduce vitrified seedlings.In particular, the production of artificial seeds does not occupy a large amount of soil. It can be produced all year round. Therefore, the research on artificial seeds has developed rapidly in the world.

Production of Artificial Seeds of Carica papaya L. var Eksotika

Artificial seed production technique for Carica papaya L. var Eksotika is considered as a valuable alternate technology of propagation. Limited availability of the elite genotypes apprehends the utility of the C. papaya L. var Eksotika plants, for industrial use and ecological improvement. Therefore, development of an efficient method for C. papaya L. var Eksotika artificial seeds production by encapsulation of micro shoots in sodium alginate matrix were produced. Artificial seeds of C. papaya L. var Eksotika were produced by encapsulated micro shoots of C. papaya L. varieties Eksotika propagated in vitro. The production of artificial plant seeds of this species offered ideal beads based upon stiffness, texture, size and shape of beads. It was found that 4% sodium alginate and harden in two hundred millimolar CaCl2.2H2O solution produced optimum beads with stiff, clear, round and homogeneous size for 30 minutes among the concentrations tested.

Artificial Seed Production of Tylophora indica for Interim Storing and Swapping of Germplasm

Our research work demonstrates the single bead alginate-encapsulation, interim storing and conversion of Tylophora indica (Burm. Fil.) Merrill. Most effective encapsulation of in vitro nodal segments [(4 ± 1) mm long], ensuing in sphere-shaped artificial seeds of similar morphology, was achieved through 75 mmol⋅L−1 calcium chloride (CaCl2 ⋅ 2H2O) plus 3% (w/v) Na-alginate with 93.3% conversion frequency. The earliest conversion (within 7 days of incubation) of artificial seeds occurred in half-strength liquid Murashige and Skoog medium. Among the three different temperature regimes [(5 ± 1) °C, (15 ± 1) °C, and (25 ± 1) °C], storage of artificial seeds at (15 ± 1) °C executed the highest frequency of conversion (90%) after 15 days of storage. Lengthier storage significantly reduced the conversion frequency of artificial seeds irrespective of storage temperature. Nevertheless, the conversion frequency after 30 days of storage at (15 ± 1) °C was recorded at 70% without further decline even following 45 days of storage, which evidently suggests that lower temperature (15 ± 1) °C is apt for storage and subsequent conversion of T. indica artificial seeds. The present protocol could be expedient for short-term storing and swapping of T. indica germplasms between national and international laboratories.

Micropropagation of Cymbidium aloifolium (L.) SW., A Medicinal Orchid by Artificial Seeds Technology

Artificial seed technology is a rapidly growing area of research in plant cell and tissue culture. Application of this technology opens an alternative route for mass scale production, efficient delivery of cloned plantlets and fulfils the increasing demand of local growers. An attempt was made to produce artificial seeds and their subsequent regeneration of a highly valuable medicinal orchid of Nepal i.e. Cymbidium aloifolium. Artificial seeds were obtained through encapsulation of protocorms in calcium alginate beads. Protocorms were encapsulated by using 3% sodium alginate and 0.2 M calcium chloride solution. Murashige and Skoog (MS) medium (1962) was used as the basal medium for in vitro germination and seedling development of artificial seed. In Cymbidium aloifolium, 20-25 days old in vitro grown protocorms were used for production of artificial seeds. Artificial seeds were inoculated on two different culture conditions of MS medium i.e. MS solid & MS liquid with four different treatments i.e. strength of 1.0, ½, ¼ and MS media supplemented with plant growth regulators viz. BAP (0.5 mg/l) and NAA (0.5 mg/l). Highest percentage of germination (100%) and plantlet conversion was found on hormone free full strength (1.0 MS) of MS liquid medium after 13-14 weeks of culture. Plantlets regenerated from artificial seeds with well developed shoot and root systems were successfully acclimatized in potting mixture of cocopeat, litter and sphagnum moss in a ratio 2:1:1.J. Nat. Hist. Mus. Vol. 28, 2014: 42-48

Selection of optimal stage for protocorm-like bodies and production of artificial seeds for direct regeneration on different media and short term storage of Dendrobium Shavin White

Micropropagation is currently the most popular method for orchid propagation through the production of protocorm-like bodies (PLBs). It is suggested that converting the PLBs into artificial seeds by encapsulation with sodium alginate can be useful for short-term preservation and distribution to the laboratories and commercial nurseries. Prior to the production of artificial seeds, the best developmental stage of PLBs based on sizes for increased conversion to plantlet was determined. PLBs were categorized based on size and presence of shoot namely ≤2 mm (S1), >2–4 mm (S2), >4–6 mm (S3), >2–4 mm with shoot (S4) and >4–6 mm with shoot (S5). S4 and S5 gave significantly higher conversion percentage (85 and 90 %, respectively) as compared to the PLBs without shoot (S1, S2 and S3). Thus, for uniformity PLBs of 3–5 mm with shoot were used for encapsulation with sodium alginate to form artificial seeds. The feasibility of germinating artificial seeds of Dendrobium Shavin White in different substrates namely; M1 (semi-solid ½ Murashige and Skoog (1962) basal medium), M2 (cotton irrigated with sterilized liquid ½ MS basal medium), M3 (cotton irrigated with sterilized distilled water) and M4 (cotton irrigated with non-sterilized distilled water) was tested. The encapsulated PLBs regenerated well in M1 where 96 % of encapsulated PLBs germinated after 12 days of inoculation and 76 % of them converted into plantlet after 37 days of inoculation while PLBs subjected to sterile distilled water gave 56 % germination and 44 % conversion after 42 and 167 days of inoculation respectively. The ability to store encapsulated PLBs would be advantageous for transport of planting materials. Encapsulated PLBs survived longer when stored at 25 ± 2 °C compared to 4 °C, 10 °C and 30 ± 2 °C whereby storage up to 75 days retained 80–92 % survival. Further storage up to 135 days retained 52 % survival. All plantlets survived after acclimatization when transferred to charcoal media under shade.

THE PRODUCTION OF CAULIFLOWER MICROSHOOTS USING CURD MERISTEMATIC TISSUES AND HYPOCOTYL-DERIVED CALLUS

The capacity for microshoot production from cauliflower was investigated applying two different protocols. In the first, cauliflower curd meristematic tissue was used as a source of explants. The meristematic layer was shaved off and the clusters produced were homogenised using a commercial blender. In terms of its effect on the number and viability of microshoots, the use of 30 s blending duration treatment was found to be optimal between several treatments tested in the range 15 to 120 s. Explants were cultivated in agitated S23 (MS (Murashige and Skoog, 1962) + 3% sucrose) liquid media supplemented with different combinations of plant growth regulators. The use of 2 mg/L kinetin and 1 mg/L IBA gave the optimal results in terms of the number and viability of microshoots. The second protocol was designed to investigate the regeneration potential of hypocotyl explants of cauliflower via callus culture. The callus tissue was initiated from hypocotyl explants in callus induction medium (CIM), which consisted of S23 supplemented with 2,4-D at 1 mg/L and kinetin at 1.5 mg/L. The highest number of shoots was obtained after 28 days from sub-cultured hypocotyl derived callus on S23 basal media containing 0.5 mg/L of kinetin. This study demonstrated the ability of producing microshoots using various parts of cauliflower through both callus and without callus formation, which can be useful in the later applications of cauliflower tissue culture such as the production of artificial seeds.

In vitro regeneration of Aristolochia tagala and production of artificial seeds

Protocols for in vitro plant multiplication from somatic tissues and production of artificial seeds through encapsulation of nodes were developed for Aristolochia tagala Cham., a rare and valuable medicinal plant, as a measure of conservation and as a prerequisite for genetic transformation procedure. A maximum number of adventitious shoots were regenerated from leaf-derived callus on Murashige and Skoog (MS) medium containing 6-benzylaminopurine (BAP; 2 μM), α-naphthaleneacetic acid (NAA; 0.5 μM), and phloroglucinol (PG; 10μM). Nodes collected from in vitro established shoot cultures were encapsulated in 3 % (m/v) sodium alginate and 1 % (m/v) calcium chloride. Multiple shoots were successfully regenerated from the encapsulated nodes cultured on MS medium supplemented with 3 μM BAP and 0.5 μM kinetin (KIN). Regenerated shoots from callus and artificial seeds were successfully rooted and acclimated to greenhouse conditions. Since roots of A. tagala are primarily used in traditional medicine, a protocol for regenerating roots directly from the leaf derived callus was also developed. Maximum root length was obtained when the callus was cultured in MS medium supplemented with KIN (1 μM), indole acetic acid (IAA; 0.5 μM), NAA (0.1 μM), and PG (10 μM). Biochemical parameters were studied in calli grown with and without PG in the medium to establish a correlation between these parameters and shoot morphogenesis. An increment of antioxidant enzymes (peroxidase and catalase) and metabolites (sugars and proteins), and a decrease in the amount of polyphenol oxidase was observed in the calli which were grown in the presence of PG.

Encapsulation of cauliflower (Brassica oleracea var botrytis) microshoots as artificial seeds and their conversion and growth in commercial substrates

An effective protocol for the mass production of cauliflower microshoots was refined using the meristematic layer of cauliflower curd. After the meristematic layer was surface sterilized and shaved off, a commercial blender was used for homogenization and several blending treatments were tested in the range 15–120 s and 30 s was found to be optimal in terms of the amount explants produced and their subsequent growth ability. Explants were cultivated in S23 liquid medium (4.4 g L−1 MS (Murashige and Skoog) and 3% v/w sucrose) supplemented with several combinations of plant growth regulators (PGRs) including 1 and 2 mg L−1 of Kinetin in combination with three types of auxins (indole butyric acid (IBA), Naphthaleneacetic acid (NAA) and Indole-3-acetic acid (IAA)), each at 1 and 2 mg L−1 concentration. The use of 2 mg L−1 Kinetin and 1 mg L−1 IBA gave the best results in terms of its effects on explant induction. Microshoots of different sizes were encapsulated in a sodium alginate matrix and the optimal stage suitable for the production of artificial seeds was assessed in terms of both subsequent conversion and plantlet viability. The feasibility of cultivating cauliflower artificial seeds in commercial substrates (compost, vermiculite, perlite and sand) irrigated with different solution mixtures including sterilized distilled water (SDW), PGRs-free S23 medium and S23 medium supplemented with Kinetin (1 and 2 mg L−1) and IBA or NAA at (1 and 2 mg L−1) was investigated. The use of 2 mg L−1 Kinetin and 2 mg L−1 NAA applied with S23 gave the optimal response with both perlite and compost. This study showed high growth capacity of cauliflower artificial seeds in commercial substrates which is considered a promising step for their direct use in vivo.

Artificial Seed Production from Encapsulated Micro Shoots of Saintpaulia ionantha Wendl. (African Violet)

Artificial seeds were produced from encapsulated micro shoots of Saintpaulia ionantha Wendl. (African violet). The production of artificial seeds of this species gave ideal beads based on firmness, texture, size and shape of beads. The percentage of germination from encapsulated micro shoots influenced by the concentrations of sodium alginate and calcium chloride (CaCl2.2H2O) used. It was found that among the concentrations tested, 3 sodium alginate and the exposure to 1.00 mM CaCl2.2H2O solution for 30 min had produced optimal beads with firm, clear, round and uniform size and suitable for handling. It was also observed that micro shoots obtained from optimization of encapsulation matrix showed the highest percentage of germination (84). Encapsulated micro shoots exposed for 30 min in 100 mM CaCl2.2H2O solution gave the optimal time of hardening process. The findings suggested that the encapsulation method for micro shoots could be used as an alternative to artificial seed derived from somatic embryos of Saintpaulia ionantha. © 2008 Asian Network for Scientific Information.

ARTIFICIAL SEED PRODUCTION IN POINTED GOURD (TRICHOSANTHES DIOICA ROXB.)

Encapsulation of nodal segments was successfully developed for pointed gourd towards the formation of artificial seed with sodium alginate. Encapsulated nodal segments (artificial seed) were cultured in MS basal medium containing different concentrations and combinations of BAP and NAA to induce germination and shoot proliferation. Highest (95%) shoot formation was obtained in MS + 1.0 mg/l BAP followed by MS + 0.5 mg/l BAP. The encapsulated nodal segments also regenerated in vitro on different substrates. Frequency of plantlet formation was low on these substrates compared to plantlet development on MS media. Among these substrates, the percentage of plantlet formation was better on moist cotton (42%) followed by filter paper (35%). The hardened plants were transferred successfully to soil in the earthen pots. The protocol for encapsulating the nodal segments for the production of artificial seeds and their subsequent regeneration is a new area of research to develop in vitro conservation strategies for pointed gourd.DOI: http://dx.doi.org/10.3329/bjpbg.v21i1.17044

Artificial Seed Production and Cultivation of the Edible Brown Alga, Sargassum Fulvellum (Turner) C. Agardh: Developing a new Species for Seaweed Cultivation in Korea

Sargassum fulvellum is a brown alga recently introduced to the seaweed cultivation industry in Korea. There is current interest in the commercial scale of aquaculture of this species. For the artificial seeding and cultivation of this alga, growth and maturation were investigated from September 2002 to August 2003. Indoor culture experiments for maturation induction were also conducted at temperatures of 5, 10, 15, 20 and 25 ∘C and irradiances of 20, 50, 80 and 100 μmol photons m−2 s−1 under 16:8 h (L:D) photoperiod. Within a given culture test range, higher temperature and irradiance levels favoured the maturation of receptacles in S. fulvellum. Using temperature and irradiance control for thalli, artificial seed production of this species could be done one month earlier than thalli matured in nature. Under natural condition, receptacle formation of the plants began in February, and the eggs were released from March to April. For mature thalli of 200 g wet wt., artificial seeding was complete enough for attachment on seed strings of 100 m. Mean production obtained from the artificial seeding technique in situ was 3.0 kg wet wt m−1 of culture rope during the cultivation period.

Effect of NaCl on in vitro propagation of sweet potato (Ipomoea batatas L.).

In vitro propagation protocols offer a better option for production of quality planting materials in a clonal crop such as sweet potato, which is a food crop of versatile uses. Propagation through axillary shoot proliferation and organogenic and embryogenic regeneration were studied in different genotypes of sweet potato. The addition of NaCl enhanced the rate of multiplication as well as yielded hardy somatic embryos. Optimal doses of NaCl in each mode of propagation were different. The hardy somatic embryos produced in NaCl medium could be stored at 8 degrees C with or without a protective alginate covering. High-frequency germination of stored hardy somatic embryos could facilitate the production of artificial seeds. Plantlets produced with the addition of NaCl in regeneration medium were established in vivo at a high frequency (95-100%). Yield and quality of storage roots developed from artificial seed-propagated plants were comparable with those of source plants.

A novel encapsulation technique for the production of artificial seeds.

A novel technique for the encapsulation of plant material in calcium alginate hollow beads was tested. The technique involves suspending plant material (i.e. plant cells, tissues, organs, shoot tips, somatic embryos) in a solution containing carboxymethylcellulose and calcium chloride and then dripping it into a stirred sodium alginate solution. In initial experiments with Daucus carota (carrot), it was found that after 14 days of cultivation, 100 % of seeds encapsulated in calcium alginate hollow beads would germinate in the liquid core and that 13% would burst the capsules. Embryogenic calli developed inside hollow beads and formed somatic embryos while calli in conventional calcium alginate beads became detached from the beads early in development, and no somatic embryogenesis occurred. With Solanum tuberosum (potato), development of calli was observed in 50% of hollow beads. Eighty-one percent of shoot tips encapsulated in hollow beads sprouted and grew out of the capsules.

Electrostatic Encapsulation and Growth of Plant Cell Cultures in Alginate

The growth of callus tissue from African Violets, encapsulated in alginate using electrostatics, was investigated as well as the mechanism of alginate droplet formation. Alginate microbeads as small as 500 (+/-50) microns in diameter could be produced by electrostatic extrusion directly from a plastic syringe (1900 micron extrusion orifice), in the absence of a needle. Video analysis of the mechanism of electrostatic alginate droplet formation from the syringe showed the development of a Taylor cone-like droplet which extended to form a thin strand that then broke up into droplets. Autoclaving of the alginate/medium solution significantly reduced its viscosity, giving smaller beads. Calculated microbead diameters agreed well with experimental values. Callus tissue from leaf explants was successfully immobilized and cultured using electrostatic extrusion. Tissue immobilized using 4% alginate in medium and cultured on agar grew best, producing a complete plantlet within four months. The long-term aim is to develop an effective method for large production of artificial seeds.

Apomixis for crop improvement

Apomixis is a genetically controlled reproductive process by which embryos and seeds develop in the ovule without female meiosis and egg cell fertilization. Apomixis produces seed progeny that are exact replicas of the mother plant. The major advantage of apomixis over sexual reproduction is the possibility to select individuals with desirable gene combinations and to propagate them as clones. In contrast to clonal propagation through somatic embryogenesis or in vitro shoot multiplication, apomixis avoids the need for costly processes, such as the production of artificial seeds and tissue culture. It simplifies the processes of commercial hybrid and cultivar production and enables a large-scale seed production economically in both seed- and vegetatively propagated crops. In vegetatively reproduced plants (e.g., potato), the main applications of apomixis are the avoidance of phytosanitary threats and the spanning of unfavorable seasons. Because of its potential for crop improvement and global agricultural production, apomixis is now receiving increasing attention from both scientific and industrial sectors. Harnessing apomixis is a major goal in applied plant genetic engineering. In this regard, efforts are focused on genetic and breeding strategies in various plant species, combined with molecular methods to analyze apomictic and sexual modes of reproduction and to identify key regulatory genes and mechanisms underlying these processes. Also, investigations on the components of apomixis, i.e., apomeiosis, parthenogenesis, and endosperm development without fertilization, genetic screens for apomictic mutants and transgenic approaches to modify sexual reproduction by using various regulatory genes are receiving a major effort. These can open new avenues for the transfer of the apomixis trait to important crop species and will have far-reaching potentials in crop improvement regarding agricultural production and the quality of the products.

High frequency production of embryos from liquid flask cultures of oilseed rape

As part of the program to scale-up the production of artificial seeds of winter oilseed rape, Brassica napus ssp. oleifera, we established a liquid flask culture system that enables the high frequency production of freely suspended embryos. As many as 4000 embryos could be obtained from 1 mL packed-cell-volume of cells. For initiation of liquid flask cultures, four different types of callus tissues were used. Among them, the most embryogenic cell suspension cultures were obtained from spontaneous callus developed on the surface of secondary embryos precultured in medium supplemented with 2,4-dichlorophenoxyacetic acid (4.52 microM) and kinetin (0.46 microM) (type B callus). Growth curves of the cell suspension were determined and the cell suspension was able to grow in medium without plant growth regulators. Embryos were observed to developed directly from the cells without going through an obvious callus phase. When subcultured to agar medium containing 44.38 microM benzylaminopurine, about 43% of the embryos developed into plants.

Artificial Seed Production of Short Neck Clam Ruditapes Philippinarum in Yamaguchi Prefecture

Artificial Seed Production of Short Neck Clam Ruditapes Philippinarum in Yamaguchi Prefecture