Frequency Of Plantlet Formation Research Articles

ФАК ТОРЫ ПРЯМОГО ПРОРАСТАНИЯ МИКРОСПОРОГЕННЫХ ЭМБРИОИДОВ BRASSICA NAPUS L.

Isolated microspore culture is the main method of producing doubled rapeseed haploids and is widely used in research institutions and commercial companies. The protocol of rapeseed embryo production is well developed and efficient for many genotypes, but some issues remain due to the low regeneration frequency of plantlets from embryos. When the standard protocol is applied, regeneration of plantlets from microspore-derived embryos usually involves a callus-forming stage followed by regeneration of adventitious shoots or secondary embryos, which prolong the period of plantlet regeneration and makes production of doubled haploids complicated. Identifying factors, which affect the frequency of direct embryo germination will increase the frequency of plantlet formation and reduce the period of DH plant production. In this work, we studied the effect of medium pH on the duration of plantlet regeneration from rapeseed microspore-derived embryos and effect of their low temperature treatment of +1 and +5℃ for 3, 6, 8, 9, 12 days in complete darkness on the embryo maturation and germination. Rising the pH of the nutrient medium from 5.8 to 6.1 increased the frequency of direct embryos germination up to 18% and the overall frequency of plantlet regeneration up to 76%. Culturing embryos at low temperatures effected the frequency of direct germination of embryos into plantlets. The maximum frequency of 44–53% direct embryo germination was observed when cultured at +1℃ for 6 and 9 days, when embryos were cultured at +5℃ the frequency of direct germination was 0–10%. In the control variant without cold treatment it was 16%.

Production of chrysanthemum synthetic seeds under non-aseptic conditions for direct transfer to commercial greenhouses

A novel protocol for both production and sowing of chrysanthemum synthetic seeds in non-aseptic conditions for large-scale commercialization was successfully established. Effects of the availability of organic compounds, namely MS vitamins and sucrose, inside and outside gelling matrix on the microbial contaminations, plantlet formation and subsequent growth were investigated. Results showed that the presence of organic compounds, either in gelling matrix alone or in both gelling matrix and commercial substrate, caused microbial contaminations in all synthetic seeds and complete inhibition in regrowth. In contrast, the absence of organic compounds in both non-sterile gelling matrix and substrate resulted in 70 % plantlet formation after 6 weeks of sowing. However, organic compounds absent in gelling matrix but present in substrate induced lower plantlet formation frequency (34 %) after 6-week sowing. The removal of organic compounds from both gelling matrix and substrate, in comparison with the removal from only gelling matrix, also stimulated the formation of plantlets having more leaves, longer shoots and roots, and greater fresh and dry biomass accumulation, but equal leaf area, chlorophyll content, and number of nodes and roots. These findings suggest that the eradication of all organic compositions is a prerequisite for practical application of encapsulation technology to the mass production of chrysanthemum plants.

EFFECT OF SINGLE-WALL CARBON NANOTUBE ON SOYBEAN (GLYCINE MAX) REGENERATION FROM MATURE COTYLEDONARY NODE EXPLANTS

One of the major limitations in producing transgenic soybeans using the agrobacterium-mediated cotyledonary-node method is low regeneration frequency. An improved highly efficient regeneration system of soybean was established herein. Cotyledonary node explants were placed in shoot initiation medium with single-wall carbon nanotube (SWNT) for adventitious shoots regeneration, and adventitious shoots were subcultured in shoot elongation medium with SWNT for shoot elongation and rooting. 40 mg/L SWNT supplemented in shoot initiation medium was found to be the optimal concentration with shoots regeneration frequency significantly increased by 21.5% compared with the control treatment, while for 4 mg/L and 400 mg/L, the increase was 4.6% and 6.5%, respectively. Faster elongation and rooting of adventitious shoots was observed in shoot elongation medium with 40 mg/L SWNT. Soybean plantlet formation frequency within the limited four weeks supplemented with 40 mg/L SWNT reached 48.4% while in the other three treatments: 4 mg/L, 400 mg/L and control, 0% were observed. These results indicate that supplement of SWNT in the soybean medium can efficiently promote adventitious shoots formation frequency, increase plantlet formation frequency and shorten the regeneration period.

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

Factors affecting plantlet regeneration from in vitro cultured immature embryos and cotyledons of Prunus mume “Xue mei”

Using immature embryos and cotyledons as explants, a successful system to culture immature embryos and induce direct regeneration from cotyledons was established for Prunus mume “Xuemei”. For immature embryo culture, a high frequency of plantlet formation (89.5%) from the embryonic axis was obtained using half-strength Murashige and Skoog (1/2 MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). Shoots formed directly from cotyledons with the embryo axis intact when explants were cultured on 1/2 MS medium containing 2.2 μM BA with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when the embryonic axis was removed from the cotyledons and cultured on 1/2 MS medium supplement with 13.2 μM BA, 2.7 μM NAA or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA, respectively. Regenerated shoots were successfully rooted on 1/2 MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of the embryonic axis, BA, and TDZ on cotyledon regeneration was investigated in detail. Rooted plantlets were transferred to soil successfully.

Somatic embryogenesis from mesocotyl and leaf-base segments of Paspalum scrobiculatum L., a minor millet

Somatic embryos could be induced from embryogenic callus originating from mesocotyl as well as leaf-base segments of Paspalum scrobiculatum on Murashige and Skoog (MS) or Chu et al. (N6) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9.0, 18.0, and 22.5 μM). N6 medium was better than MS, for both explants, for high-frequency somatic embryogenesis. Also, mesocotyl tissues were relatively more totipotent than leaf-base segments. The somatic embryos ‘germinated’ and formed plantlets on transfer of embryogenic calluses to hormone-free MS or N6 regeneration medium. Embryogenic cultures could be maintained on low hormone medium which readily regenerated to form plantlets on hormone-free medium. A higher frequency of plantlet formation occurred on MS than on N6 medium. In vitro-formed plantlets were gradually acclimatized in the culture room and on transfer to soil flowered and set seed. Somatic embryogenesis and plantlet regeneration from mesocotyl and leaf-base segments are potentially simpler systems than regeneration from ‘embryonic’ explants such as immature embryos and unemerged inflorescences.

Efficient regeneration from GUS-transformed Ajuga hairy root

A procedure for the systematic regeneration from Ajuga hairy root was developed. Supplementation of an auxin, naphthaleneacetic acid (0.1 mg/ l), in the hairy root growth culture enhanced the cell growth rate. Mechanical fragmentation for 10 s in a commercial blender enabled a large number of root fragments to be produced efficiently. Supplementation of a cytokinin, benzyladenine (10 mg/ l), exhibited the highest plantlet formation frequency in the plantlet formation stage. The plantlets longer than 4 mm in length were able to grow into whole plants on agar containing modified MS medium. Ajuga hairy root in which the β-glucuronidase (GUS) gene was introduced under control of the tomato ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit ( rbcS3B) promoter was constructed by the Agrobacterium rhizogenes-mediated co-transformation. The GUS-transformed hairy root could also be efficiently regenerated into plantlets through this procedure. GUS activity was detected in leaf tissue of the regenerated plants.

Micropropagation of horseradish hairy root by means of adventitious shoot primordia

Adventitious shoot primordia were formed on horseradish hairy root cultured in dark. Plantlet formation frequency from the primordia was higher than that from root fragments. Culture for 26 days provided the adventitious shoot primordia, which had the highest potential for plantlet formation (53% explants at 40 days). Benzyladenine supplementation in the dark caused primordium enlargement, but did not increase the number of primordia formed. After adventitious shoot primordia were encapsulated with calcium alginate, kinetin supplementation (2.0–4.0 μM) increased the shoot formation frequency (65–80% explants at 20 days) in the light, but also promoted the undesirable formattion of multiple shoots. Supplementation with naphthaleneacetic acid (0.27–5.4 μM) in the calcium alginate beads in light enhanced the root emergence from primordia without inhibition of plantlet formation when the encapsulated beads were put on the agar-medium without naphthaleneacetic acid.

Studies on the Mechanisms of Pollen Embryogenesis VIII. The Role of Na2 EDTA in Pollen-plant Formation of Nicotiana tabacum L.

The effects of mineral salts on plantlet formation were investigated in tobacco anther culture paying special attention to Na2 EDTA, a component of FeEDTA.Removal of mineral salts other than FeEDTA from the basal medium had little influence on plantlet formation. While, removal of FeEDTA arrested plantlet formation severely, but did not affect the induction of embryogenic pollen grains. Moreover, very few plantlets were obtained from anthers transferred to FeEDTA-free medium after 3 weeks of culture. From these results, it is concluded that FeEDTA plays the most important role in the development from embryoid to green plantlet.Plantlet formation was almost completely arrested on Na2EDTA-free medium. In contrast, plantlet formation on ferrous sulphate free (Na2EDTA) medium was comparable to that on basal medium. Also, the highest frequency of plantlet formation was obtained at the same concentration (2×10-4M) for both FeEDTA and Na2 EDTA. These results suggest that the effects of FeEDTA on the plantlet formation in tobacco anther culture refer to the function of Na2EDTA itself.

Promotion of plantlet formation from somatic embryos of carrot treated with a high molecular weight extract from a marine cyanobacterium

Somatic embryos of Daucus carota L. developed into plantlets at high frequency after addition of an extract from a marine cyanobacterium, Synechococcus sp. NKBG 042902. High molecular weight, nondialyzing fraction, separated from the extract, possessed enhanced plantlet formation promoting activity. Plantlet formation frequency was 60 % after addition of nondialysate (100 mg/l) compared to 28 % without addition. Embryos treated with the nondialysate contained five times more chlorophyll than nontreated embryos after 6 days of culture. The chlorophyll a/b ratio of 4-day old treated somatic embryos was found to be similar to that of zygotic embryos. However, the chlorophyll a/b ratio of plantlets induced from nontreated somatic embryos was variable. Nondialysate was fractionated by ultracentrifugation and an active component obtained, which gave a maximum plantlet formation frequency of 71 %, and induced rapid greening of shoots.

Regeneration of Cucumis sativus var. sativus and C. sativus var. hardwickii, C. melo, and C. metuliferus from explants through somatic embryogenesis and organogenesis

Regeneration in six inbred lines or F1 hybrids of Cucumis sativus was achieved on Murashige & Skoog's medium containing various concentrations of 2,4-D/BA, NAA/BA, NAA/Z or NAA/K. The range of regeneration frequency for cotyledon, leaf and petiole explants was 0–38, 0–75 and 14–96%, respectively, after 6–8 weeks in culture. Only one subculture of calli to growth regulator-free medium was required for regeneration. Preincubation of explants in the dark for 2–3 weeks was essential to achieve optimal regeneration. Highest frequency of plantlet formation occurred with petiole explants incubated on NAA/BA (5.0/2.5 μM), NAA/Z (5.0/5.0 μM) or 2,4-D/BA (5.0/5.0 μM). Approximately 80% of these plantlets survived after transplanting to greenhouse soil, and they flowered and set fruit. The F1 hybrid, Endeavor, gave the highest regeneration frequency of 91% on 2,4-D/BA at 5.0/5.0 μM. Formation of somatic embryos was observed on 2,4-D/BA, while organogenesis and embryogenesis both were evident on NAA/BA and NAA/Z. Cotyledonary explants yielded the lowest frequency (ca. 7%) of plantlet formation in this study. Plantlets of C. sativus var. hardwickii and an F1 hybrid of C. sativus x C. s. var hardwickii were regenerated on NAA/Z and NAA/K at frequencies of 15–65%, predominantly by the formation of somatic embryos. Shoots were obtained from cotyledon and leaf explants of C. metuliferus on IAA/BA (7.5/5.0 μM) and from leaf and petiole explants of C. melo on NAA/BA (5.0/2.5 μM), but plantlets were recovered only in C. melo.

Combining abilities and heritability of callus formation and plantlet regeneration in wheat (Triticum aestivum L.) anther cultures

Frequency of callus formation in wheat (Triticum aestivum L. em. Thell) anthers cultured in vitro and the frequency of subsequent plantlet formation from such calli were examined in a diallel population produced from five inbred spring wheat cultivars. Two of the five cultivars were believed to possess relatively high frequencies of response and the other three relatively low response frequencies, based on previous studies. General and specific combining abilities were estimated and found to be highly significant for both traits. Reciprocal effects were also estimated and were highly significant for both traits. Of the 25 entries, the largest mean callus formation frequency was observed on anthers of 'Kitt' x 'Olaf', while the largest mean plantlet formation frequency was observed using anthers of the cultivar, 'Fielder'. No significant correlation was observed between the two traits. Heritability estimates in the range of 0.6-0.7 suggested, however, that both traits were highly heritable, so that rapid gain from selection for these traits should be possible. Current limitations due to genetic variation in responses therefore may not constitute a major obstacle to application of in vitro techniques by wheat breeders.

Relationships Between Anther Browning and Plantlet Formation in Anther Culture of Nicotiana tabacum L.

Summary Anthers of Nicotiana tabacum cv. Wisconsin 38 contammg pollen grains in various developmental stages were cultured for 55 days at 23°C to investigate the relationship between the formation of plantlets from pollen and browning of the anther. Browning took place in anthers containing binucleate pollen grains during the first 10 days after inoculation onto Nitsch's basal medium (1969) , whereas it was continuously observed for as long as a month in anthers containing micros pores in the uninucleate stage and in those undergoing pollen mitosis. Plantlet formation in the first instance was observed during the first 3 to 5 weeks of culture and was never detected thereafter. There was no positive correlation between anther browning and plantlet formation. Plantlet induction was delayed for one week, but it was observed to be continuously effective until the cultures were 50 days old in the anthers containing uninucleate and mitotic microspores. The protraction of senescence in anthers appears to be of great importance for the induction of embryogenesis in microspores at this stage of development. There was a positive correlation between the duration of anther browning and the time required for plantlet production. That is, the percentage of plantlet formation in anthers having browned during the first 2 weeks was low, whereas the frequency of plantlet formation was high in long-lived anthers. These results suggest that there is a critical period for the induction of pollen embryogenesis in the early binucleate stage. Most microspores not yet having undergone pollen mitosis can attain to this stage with the nutritional or hormonal aid of living anther tissue; the embryogenesis can never proceed if anther browning has occurred before this stage has been attained. In a more advanced stage the duration of such a situation seems to be shorter.