共查询到20条相似文献,搜索用时 245 毫秒
1.
Wen Liu Xuesen Chen Guanjun Liu Qing Liang Tianming He Jianrong Feng 《Plant Cell, Tissue and Organ Culture》2007,88(3):289-299
Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th
week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue
such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l−1 6-BA and 0.5 mg l−1 IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l−1 6-BA and 1.0 mg l−1 IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l−1 IAA and 0.2 mg l−1 NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple
sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility,
and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also
increased the multiplication coefficient of embryo-induced shoots. 相似文献
2.
Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and
pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated
in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division
occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred
within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest
with modified MS medium in which NH4NO3 was replaced with 3.0 g l−1 glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts
isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing
0.5 mg l−1 2,4-D + 1.0 mg l−1 kinetin or 2.0 mg l−1 BAP + 1.0 mg l−1 dicamba + 0.1 mg l−1 NAA + 80 mg l−1 adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l−1 2,4-D + 1.0 mg l−1 kinetin or 1.0 mg l−1 kinetin + 0.5 mg l−1 GA3 + 80.0 mg l−1 adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later
embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased
amounts of DNA in about one third of the regenerants. 相似文献
3.
Karin Sonntag Brigitte Ruge-Wehling Peter Wehling 《Plant Cell, Tissue and Organ Culture》2009,96(3):297-305
A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 105 protoplasts g−1 fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all
tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks)
and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown
pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids.
However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast
fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration. 相似文献
4.
Yulin Cui Jinfeng Wang Peng Jiang Shuguang Bian Song Qin 《World journal of microbiology & biotechnology》2010,26(9):1653-1657
A transient transformation system for the unicellular marine green alga, Platymonas subcordiformis, was established in this study. We introduced the pEGFP-N1 vector into P. subcordiformis with a glass bead method. P.
subcordiformis was incubated in cell wall lytic enzymes (abalone acetone powder and cellulase solutions) to degrade the cell wall. The applicable
conditions for production of viable protoplasts were pH 6.5, 25°C, and 3 h of enzyme treatment. The protoplast yield was 61.2%
when P. subcordiformis cells were added to the enzyme solution at a concentration of 107 cell ml−1. The protoplasts were immediately transformed with the pEGFP-N1 vector using glass-bead method. The transformation frequency
was about 10−5, and there was no GFP activity observed in either the negative or the blank controls. This study indicated that GFP was a
sensitively transgenic reporter for P. subcordiformis, and the method of cell wall enzymolysis followed by glass bead agitation was applicable for the transformation of P. subcordiformis. 相似文献
5.
Vishal Gupta Manoj Kumar Puja Kumari C. R. K. Reddy Bhavanath Jha 《Journal of applied phycology》2011,23(2):209-218
This study reports on the optimization of protoplast yield from two important tropical agarophytes Gracilaria dura and Gracilaria verrucosa using different cell-wall-degrading enzymes obtained from commercial sources. The conditions for achieving the highest protoplast
yield was investigated by optimizing key parameters such as enzyme combinations and their concentrations, duration of enzyme
treatment, enzyme pH, mannitol concentration, and temperature. The significance of each key parameter was also further validated
using the statistical central composite design. The enzyme composition with 4% cellulase Onozuka R-10, 2% macerozyme R-10,
0.5% pectolyase, and 100 U agarase, 0.4 M mannitol in seawater (30‰) adjusted to pH 7.5 produced the highest protoplast yields
of 3.7 ± 0.7 × 106 cells g−1 fresh wt for G. dura and 1.2 ± 0.78 × 106 cells g−1 fresh wt for G. verrucosa when incubated at 25°C for 4–6 h duration. The young growing tips maximally released the protoplasts having a size of 7–15 μm
in G. dura and 15–25 μm in G. verrucosa, mostly from epidermal and upper cortical regions. A few large-size protoplasts of 25–35 μm, presumably from cortical region,
were also observed in G. verrucosa. 相似文献
6.
Protoplasts isolated from wild cotton Gossypium davidsonii were cultured in KM8P medium supplemented with different phytohormones. The most effective combination was 0.45 μM 2,4-dichlorophenoxyacetic acid,
2.68 μM α-naphthaleneacetic acid and 0.93 μM kinetin and the division percentage at the 8th day was 30.78 ± 3.04 %. The density of protoplasts at 2–10 × 105 cm−3 was suitable for protoplast division and calli formation, with a division percentage of 32.21 ± 3.64 % and a plating efficiency
of 9.12 ± 2.61 % at the 40th day. The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol. Protoplasts were
cultured in three ways, a double-layer culture system, with liquid over solid medium was proved to be the best way. Embryo
induction was further increased by addition of 0.14 μM gibberellic acid. 相似文献
7.
Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm−3 of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic.
Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within
two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer
to medium with gibberellic acid (GA3, 1.0 mg dm− 3) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm−3 BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots.
Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm−3 BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages. 相似文献
8.
Summary Protoplasts were isolated from Agrobacterium rhizogenes A4-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×106 per g fresh weight) was obtained from 12-d-old calluses after being subeultured on fresh medium. The viability of protoplasts
reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented
with 2 mgl−1 (9.05 μM) 2,4-dichlorophenoxyacetic acid, 0,2mgl−1 (0.93 μM) kinetin, 0.3 M mannitol, 2% (w/v) sucrose, and 500 mgl−1 casein hydrolyzate at a plating density of 1.0×105 per ml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency
was about 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without growth regulators.
Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast
culture system would be valuable for further somatic hybridization in forage legumes. 相似文献
9.
Summary Carbohydrate type and concentration and their interactive effects on in vitro shoot proliferation of three lingonberry (Vaccinium vitis-idaea ssp. vitis-idaea L.) cultivars (‘Regal’, ‘Splendor’, and ‘Erntedank’) and two V. vitis-idaea ssp. minus (Lodd) clones (‘NL1’ and ‘NL2’) were studied. Nodal explants were grown in vitro on medium with 2 μM zeatin and either glucose, sorbitol, or sucrose at a concentration of 0, 10, 20, or 30 gl−1. The interactive effects of carbohydrate type and concentration and genotype were important for shoot proliferation. The
best response was afforded by sucrose at 20 gl−1 both in terms of explant response and shoot developing potential, although glucose supported shoot growth equally well, and
in ‘NL1’ at 10 gl−1 it resulted in better in vitro growth than sucrose. Carbohydrate concentration had little effect on shoot vigor. The genotypes differed in terms of shoots
per explant, length, and vigor, leaves per shoot, and callus formation at the base of explants; this was manifested with various
types and concentrations of carbohydrate. Changing the positioning of explants on the medium from vertically upright to horizontal
increased the shoot and callus size, but decreased shoot height and leaves per shoot. Proliferated shoots were rooted on a
peat:perlite (1∶1, v/v) medium and the plantlets were acclimatized and eventually established in the greenhouse. 相似文献
10.
An efficient regeneration protocol via somatic embryogenesis was optimized for mung bean [Vigna radiata (L.) Wilczek; cv. Vamban 1]. Primary leaf explants were used for embryogenic callus induction in MMS medium (Murashige and
Skoog salts with B5 vitamins) containing 2.0 mg dm−3 2,4-dichlorophenoxyacetic acid (2,4-D), 150 mg dm−3 glutamine and 3 % sucrose. Fast growing, highly embryogenic cell suspensions were established from 21-d-old calli in MMS
medium supplemented with 0.5 mg dm−3 2,4-D and 50 mg dm−3 proline (Pro), and maximum recovery of globular (39.0 %), heart-shaped (26.3 %) and torpedo-stage (21.0 %) somatic embryos
were observed in this medium. Mature cotyledonary-stage somatic embryos were cultured for 5 d in half strength B5 liquid medium
containing 0.05 mg dm−3 2,4-D, 20 mg dm−3 Pro, 5 μM abscisic acid, 1000 mg dm−3 KNO3, 50 mg dm−3 polyethylene glycol (PEG 6000) and 30 g dm−3 D-mannitol. Mature somatic embryos were germinated after dessication for 3 d and complete development of plantlets accomplished
in MMS medium containing 30 g dm−3 maltose, 0.5 mg dm−3 benzyladenine and 500 mg dm−3 KNO3. Profuse lateral roots, and regeneration frequency (up to 60 %) were observed in half-strength MMS medium containing 0.5
mg dm−3 indolebutyric acid (IBA). The regenerated plants were grown to fruiting and were morphologically normal and fertile. 相似文献
11.
Protoplasts isolated from the mycobiont of a cultured lichen Usnea ghattensis were fused with protoplasts of the fungus Aspergillus nidulans in order to increase the growth rate of the cultured lichen mycobiont in vitro. The maximum protoplast yield (102 × 104/g fresh cell mass) was reached in citrate buffer with 50 mmol/L 2-sulfanylethanol (‘2-mercaptoethanol’) containing 0.1 %
Novozym after 1.5 h at pH 5 and ≤25 °C. The increase in the concentration of the above effectors or the addition of others
(e.g., MgSO4) as well as increase in time, shaking frequency, etc. caused the lower yield of protoplasts. The fused protoplasts were regenerated after transfer to malt extract-yeast extract
medium and produced, after a 45-d cultivation, a fresh cell mass of 0.232 g (from starting 0.3 g) along with the lichen substance
usnic acid. 相似文献
12.
Summary
Lilium Asiatic hybrid ‘Mona’ bulblets were cultured in vitro for 100 d under photoautotrophic (CO2-enriched conditions and without sucrose in the medium) and heterotrophic (non-enriched CO2 conditions and sucrose-supplemented medium) methods and under various levels of photosynthetic photon flux (PPF). Bulblet
growth and net photosynthetic rate (NPR) were analyzed. CO2− and PPF-enriched conditions enhanced the overall growth of bulblets, scale leaves, and roots. Heterotrophic conditions enhanced
bulblet growth but higher PPF levels were inhibitory to the development of scale leaves. These results indicate the CO2− and PPF-enriched conditions (photoautotrophic conditions) are beneficial for the production of high-quality bulblets of Asiatic
hybrid lilies in vitro 相似文献
13.
A micropropagation protocol through multiple shoot formation was developed for Thlaspi caerulescens L., one of the most important heavy metals hyperaccumulator plants. In vitro seed-derived young seedlings were used for the initiation of multiple shoots on Murashige and Skoog (MS) medium with combinations
of benzylaminopurine (BA; 0.5–1.0 mg dm−3), naphthaleneacetic acid (NAA; 0–0.2 mg dm−3), gibberellic acid (GA3; 0–1.0 mg dm−3) and riboflavin (0–3.0 mg dm−3). The maximum number of shoots was developed on medium containing 1.0 mg dm−3 BA and 0.2 mg dm−3 NAA. GA3 (0.5 mg dm−3) in combination with BA significantly increased shoot length. In view of shoot numbers, shoot length and further rooting
rate, the best combination was 1.0 mg dm−3 BA + 0.5 mg dm−3 GA3 + 1.0 mg dm−3 riboflavin. Well-developed shoots (35–50 mm) were successfully rooted at approximately 95 % on MS medium containing 20 g
dm−3 sucrose, 8 g dm−3 agar and 1.0 mg dm−3 indolebutyric acid. Almost all in vitro plantlets survived when transferred to pots. 相似文献
14.
Elena Palomo-Ríos Araceli Barceló-Muñoz José A. Mercado Fernando Pliego-Alfaro 《Plant Cell, Tissue and Organ Culture》2012,109(2):201-211
Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of
avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for
selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l−1 kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l−1 partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth.
For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l−1 kanamycin and 250 mg l−1 timentin for 2 months. Then, kanamycin level was increased to 100 mg l−1 for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant
calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was
confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic
embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA3 for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l−1 agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar
to control plants. 相似文献
15.
A genetic transformation system has been developed for callus cells of Crataegus
aronia using Agrobacterium
tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with
5 mg l−1 Indole-3-butyric acid (IBA) and 0.5 mg l−1 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different
types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red
colored callus was obtained on MS medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli
were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l−1 hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this
is the first time to report an Agrobacterium-mediated transformation system in Crataegus
aronia. 相似文献
16.
Xingbin Xie Jing Zhao Yu-Jin Hao Congbing Fang Yan Wang 《Plant Cell, Tissue and Organ Culture》2017,130(1):183-196
Two direct DNA transfer methods, biolistic transformation and a protoplast transformation approach using the INRA-clone 717 1B4 (Populus tremula?×?P. alba), are applied to poplars and compared. Both the in vitro culture and the transformation parameters were optimized to receive a maximum quantity of transformed cells to achieve a stable transformation. For the first time, the stable integration of gfp and dsred in the poplar genome and their expression as visual reporter genes in regenerated plantlets can be shown. For biolistic transformation, stem segments cut lengthwise and incubated for 10 days on a callus induction medium revealed the highest number of transient Gfp- and dsRed signals. After optimization of the in vitro culture parameter, Gfp and dsRed-expressing transgenic poplars were regenerated, proven by PCR and Southern blot analysis. For protoplast transformation, the focus was initially on the development of a highly efficient protoplast isolation and plant regeneration system. Using an enzyme solution consisting of 1.0% cellulase R10 and 0.24% macerozyme, 1?×?107 protoplasts were obtained from 1 g fresh weight leaves. Following incubation of the protoplasts in 600 mOsm culture medium, a high number of microcalli were obtained, from which plantlets were regenerated. The parameters for isolation and regeneration were then complemented by an efficient protoplast transformation protocol with 40% PEG1500. The results of this study confirm that both the biolistic and the protoplast transformation methods can be considered suitable for transferring cisgenes directly into poplar. 相似文献
17.
Branka Vinterhalter Dijana Krstić Milošević Teodora Janković Jelena Milojević Dragan Vinterhalter 《Central European Journal of Biology》2012,7(4):690-697
Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l−1 BA and 0.1 mg l−1 NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous
rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased
mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l−1 IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based
substrate. 相似文献
18.
Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic
callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l−1 (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l−1 (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus
had high levels of Na+, sugar, free amino acids, and proline but a slight decline was recorded in K+ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0
mg l−1 (9.0 μM) 2,4-D+0.5 mg l−1 (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l−1 (0.1 μM) α-naphthaleneacetic acid +0.1 mg l−1 (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing
0.2% (w/w) NaCl. 相似文献
19.
In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants
was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented
with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 100 mg dm−3 glutamine, 20.9 μM N
6-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing
2 % (m/v) sucrose, 100 mg dm−3 myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis
from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing
3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular
shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous
proline content up to 28th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium
with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine. 相似文献
20.
Protoplasts were isolated enzymatically from the carrageenophyte red alga Grateloupia turuturu (Halymeniales, Rhodophyta) that occurs along the coast of the French Channel in Normandy. Effects of the main factors on
the protoplast yield were identified to improve the isolation protocol. The optimal enzyme composition for cell wall digestion
and protoplast viability consisted of 2% cellulase Onozuka R-10, 0.5% macerozyme R-10, 2% crude extract from viscera of Haliotis tuberculata, 0.8 M mannitol, 20 mM sodium citrate, 0.3% bovine serum albumin at 25°C, and 4-h incubation period. The protoplasts were
approximately 5–15 μm in diameter, liberated mainly from the surface cell layers. Maximum yield was 1.5 × 107 protoplasts g-1 fresh tissue. The protoplasts underwent initial division after 14 days with a high density level of 1 × 106 cells mL-1 in culture medium and developed into microthalli of a line of two to six cells. 相似文献