Improved Taxol production by combination of inducing factors in suspension cell culture of Taxus baccata

To date enormous attempts have been devoted to improve Taxol production exploiting various methodologies from bioprocess engineering to biotechnological and synthetic approaches. We have developed a 2-stage suspension cell culture of Taxus baccata L. using modified B5 medium in order to improve cell growth as well as productivity. After callus induction and cell line selection, B5 medium was supplemented with vanadyl sulfate (0.1 mg/l), silver nitrate (0.3 mg/l) and cobalt chloride (0.25 mg/l) at the first day of stage I culture to maximize cell growth. This medium was further supplemented with sucrose (1%) and ammonium citrate (50 mg/l) on day 10 and sucrose (1%) and phenylalanine (0.1 mM) on day 20 (i.e., biomass growth medium). At stage II (day 25), two different concentrations of several elicitors such as methyl jasmonate (10 or 20 mg/l), salicylic acid (50 or 100 mg/l) and fungal elicitor (25 or 50 mg/l) were added to the biomass growth medium with the aim of improving cellular productivity. For morphological analysis, microscopic inspection was carried out during cultivation. Cell-associated and extracellular amount of Taxol were detected and measured using HPLC methodology. At stage I, overall Taxol amount of biomass growth medium was 13.75 mg/l (i.e., 5.6-fold higher than that of untreated B5 control). At stage II, treated cells with methyl jasmonate (10 mg/l), salicylic acid (100 mg/l) and fungal elicitor (25 mg/l) produced the highest amount of Taxol (39.5 mg/l), which is 16-fold higher than that of untreated B5 control (2.45 mg/l). Microscopic analyses of Taxus cells in suspension cultures showed various positional auto-fluorescence showing direct correlation with Taxol production. Our studies revealed that intervallic supplementation of B5 medium with combination of biomass growth factors at stage I and mixture of elicitors at stage II could significantly increase Taxol production. Thus, we suggest that the exploitation of this methodology may improve the production of Taxol since demands for Taxol pharmaceuticals are increasingly growing and resource paucities have limited its direct harvesting from Taxus trees.     

Culture conditions for growth and solvent biosynthesis by a modified Clostridium acetobutylicum

Summary A modified strain of Clostridium acetobutylicum and the fermentation medium conditions for good growth of the culture and normal production of solvents are described. The pretreatment of the culture with butyric-acid-enriched medium increased the final solvent yield on sugar and lowered the residual butyric acid accumulation. In a complex medium, relatively high concentrations of yeast extract (7.5 g/l) and ammonium sulphate (3 g/l to 6 g/l) were required for normal solvent synthesis. The nitrogen requirements for cellular growth and solvent production were distinctively different. Production of solvents and growth of the culture were dependent on the concentration of para-aminobenzoic acid and relatively independent of the variations of the initial pH of the medium in the range of 4.6 to 6.3. Solvent production was obtained with initial glucose concentrations of 20.5 g/l to 70 g/l, resulting in a maximum solvent concentration of 22 g/l and a maximum yield on glucose of 32.7%.     

Influence of growth conditions on production of capsular and extracellular polysaccharides byRhizobium leguminosarum

The influence of growth rate and medium composition on exopolymer production byRhizobium leguminosarum was studied. When grown in medium containing 10g/l mannitol and 1g/l glutamic acid,Rhizobium leguminosarum biovartrifolii TA-1 synthesized up to 2.0g/l of extracellular polysaccharide (EPS), and up to 1.6g/l of capsular polysaccharide (CPS). Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1g/l, while EPS-production remained relatively low (0.8g/l). Maximal CPS-yield was 2.9g CPS/l medium in a medium containing 20g/l mannitol and 2g/l glutamic acid. TheEPS-deficient strain R. leguminosarum RBL5515,exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets. With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500mg cell protein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h^–1). The production rates were 50–100mg/g protein/h for EPS and 15–20mg/g protein/h for CPS. Only low amounts of cyclic -(1,2)-glucans were excreted (10–30 mg/l) over the entire range of growth rates.Abbreviations bv biovar - CPS capsular polysaccharide - EPS extracellular polysaccharide - HM_r high molecular mass - LM_r low molecular mass - YEMCR Yeast Extract-Mannitol-Congo Red agar     

The response of small vessel endothelial cells from fetal rat lung to growth factors

Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.     

Production of Virus-tree Nucellar Plantlets from Unfertilized Ovules of Citrus in vitro

On a series of Murashige and Tucker (MT) media supplemented with different growth regulators, the 8-week-old unfertilized ovules of Washington navel orange (Citrus sinensis) were able to regenerate perfect plantlets via somatic embryogenesis or organogenesis. The sorts and combinations of exogenous hormones had remarkable effects on the induction, growth and differentiation of its callus. It was found that the most suitable induction medium was MT medium supplemented with 1.0 mg/l BA and 0.5mg/l 1AA. The most suitable differentiation medium was MT medium supplemented with 1.0 mg/l IBA. It was proved by indicator plant examination that the nucellar plantlets free of citrus exocortis virus (CEV) and citrus tristeza virus (CTV) had been obtained from infected trees.     

Production of solasodine by Solanum laciniatum using plant tissue culture technique

Leaf and hypocotyl explants of 15 days old aseptically grown seedlings of Solanum laciniatum were cultured on MS medium supplemented with NAA (2 mg/l) and kinetin (0.5 mg/l) for callus initiation. For maintenance and proliferation of callus MS medium supplemented with 2,4-D (1 mg/l) and kinetin (0.5 mg/l) was used. The growth of the calli derived from hypocotyls increased with time of incubation and remained almost constant after 45 days. The solasodine content in callus culture was maximum after 30 days of incubation. Addition of L-arginine in the medium (50-150 mg/l) increased growth as well as chlorophyll content in the callus culture. The solasodine content also increased up to 1.2 to 1.4 times in these cultures. High frequency shoot regeneration was obtained in MS medium having BA (4 mg/l) and IBA (0.25 mg/l). For shoot multiplication, MS medium having BA (4 mg/l) was used. Shoots rooted on the same medium. Organogenesis promoted solasodine accumulation in the cultures. Regenerated shoots yielded higher solasodine content than undifferentiated as well as organogenic callus. Solasodine contents in the regenerated shoots was found to be 10 times higher than the callus culture and approached towards the field grown plants. Thin layer chromatography revealed the presence of three compounds. The most predominant spot (Rf 0.789) corresponded to the reference solasodine.     

不同因素对岩黄连悬浮细胞生长的影响

目的:建立一个快速生长的岩黄连悬浮细胞培养体系。方法:研究了接种量、基本培养基、初始pH值、不同碳源对岩黄连悬浮细胞生长的影响。结果:合适的接种量是7.5~10%(FW),接种量过少会抑制细胞生长;B5和MS基本培养基均适合岩黄连细胞的生长;最佳初始培养基pH值为6.0,此时获得的细胞生物量最高;岩黄连悬浮细胞培养的生长周期为24d,最大生物量出现在第18d,达到14.1g/l(DW);蔗糖比葡萄糖更有利于岩黄连细胞的生长,添加60g/l蔗糖所获得的生物量最高,达到18.5g/l(DW)。     

Impact of paper bridges, activated charcoal, and antioxidants on growth and development of protocorm-like bodies of hybrid Cymbidium

This study aimed to establish whether protocorm-like bodies (PLBs) of hybrid Cymbidium Twilight Moon ‘Day Light’ could be cultured on paper bridges to remove tissue and medium browning and enhance growth. In addition, the effects of two antioxidants (ascorbic acid [AA] and α-tocopherol [AT]) and activated charcoal (AC) on tissue and medium browning were evaluated. Half-PLBs were cultured on Whatman No. 1 paper bridges in 10 ml of medium containing AC, AA, or AT. The production and development of new PLBs was inferior to that on standard agar-based solid medium when half-PLBs were used. The addition of 1 g/l AC, 10 mg/l AA, or 25 mg/l AT did not affect the formation of new PLBs from half-PLBs, but tissue or medium browning was also not observed. Encapsulated PLBs developed from half-PLBs germinated on medium containing AC or antioxidants. This research provides a simple but effective chemical means, through the use of AC or antioxidants, of avoiding tissue or medium browning without negatively impacting the growth and productivity of orchid PLBs. The use of paper bridges, however, significantly reduced PLB-related growth and development parameters relative to agar-based medium.     

Direct somatic embryogenesis and plant regeneration from single cell suspension cultures of Elymus giganteus Vahl

Summary A yellowish, nodular callus was induced from mature embryos of Elymus giganteus Vahl on MS medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l kinetin, from which a cell suspension culture was initiated in liquid MS medium supplemented with 0.5 mg/l 2,4-D, 1.0 mg/l kinetin and 0.2 mg/1 naphthaleneacetic acid (NAA). By filtering through a series of sieves with decreasing mesh sizes and collecting the resultant filtrate, a suspension culture composed mainly of single embryogenic cells was established. In a medium containing 0.3 mg/l 2,4-D, 1.0 mg/l 6-benzylaminopurine (6-BAP) and 500 mg/l casein hydrolysate (CH), the single cells underwent direct somatic embryogenesis resulting in the formation of proembryos. These proembryos developed into mature embryos when placed in a double-layer liquid overlay culture. Intact plants were developed from somatic embryos when they were transferred onto solidified MS medium without added growth regulators.     

Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Primula obconica

Protoplasts were isolated from cell suspension cultures of Primula malacoides cv. ‘Lovely Tokyo’ and P. obconica cv. ‘Aalsmeer Giant White’. P. obconica protoplasts were embedded in 0.1% (w/v) gellan gum-solidified discs comprising MS medium supplemented with 3 mg/l of 2,4-D or picloram, 0.1 mg/l of zeatin, 0.2 M glucose and 0.2 M mannitol, and surrounded by a liquid medium of the same composition except for the addition of 0.1% (w/v) activated charcoal. The protoplasts formed visible colonies, which were transferred to the regeneration medium containing 30 g/l of sucrose, 0.1 mg/l of picloram and 2 mg/l of zeatin for shoot induction. P. malacoides protoplasts formed visible colonies when cultured in disc culture using 0.1% (w/v) gellan gum-solidified MS medium containing 5 mg/l of 2,4-D, 1 mg/l of NAA, 0.1 mg/l of zeatin and 0.4 M glucose. Small calli were transferred to MS medium supplemented with 5 mg/l of zeatin for shoot regeneration. The shoots of both species readily rooted on plant growth regulator-free 1/2 MS medium and successfully acclimatized to greenhouse conditions. The protoplast-derived plants showed some alterations in morphological characteristics from those of the in-vitro-germinated control plants.     

Micropropagation of the Thai orchid <Emphasis Type="Italic">Grammatophyllum speciosum</Emphasis> blume

Protocorm-like bodies (PLBs) were induced from shoot tips of Grammatophyllum speciosum, a Thai orchid. The highest frequency of PLBs (93%) were observed on explants incubated on 1/2-Murashige and Skoog (MS) liquid medium containing 2% (w/v) sucrose without any plant growth regulators (PGRs). Tests with different carbon sources compared to sucrose revealed that maltose promoted the highest relative growth of G. speciosum PLBs (7-fold increase), while trehalose and sucrose yielded 5-fold and 4-fold increases, respectively. In 1/2 MS liquid medium, addition of 15 mg/l of chitosan promoted a 7-fold increase in PLB growth while 25 mg/l promoted a 4-fold increase. However, the relative growth rate in solid culture was significantly lower than that in liquid culture. In addition, chitosan supplementation in solid medium promoted shoot formation but not rooting. Plantlet regeneration was induced using a combination of NAA and BA supplementation in 1/2 MS solid medium with optimum induction shoot and root formation at 2.0 mg/l NAA and 1.0 mg/l BA. Using this protocol, approximately 8 months was required to obtain a hundred plantlets from one shoot tip. The plantlets showed no changes in ploidy when tested by flow cytometry.     

Transformation of Solanum nigrum L. protoplasts by Agrobacterium rhizogenes

Summary Solanum nigrum protoplasts were co-cultivated with Agrobacterium rhizogenes harboring agropine-type Ri plasmid (pRi15834). A large number of transformed calli were obtained on Murashige and Shoog's (MS) medium lacking plant growth regulators. Frequency of transformation was about 3.5×10^–3. In most of the calli, hairy roots appeared on MS medium without plant growth regulator. When the hairy roots were cut into segments and subcultured on MS medium lacking plant growth regulators, calli were readily formed. Plantlets were regenerated by transferring those calli to MS medium supplemented with 1 mg/l zeatin and 0.2 mg/l naphthaleneacetic acid. Frequency of plant regeneration was about 70%.     

Plant regeneration from leaf protoplasts of <Emphasis Type="Italic">Solanum virginianum</Emphasis> L. (<Emphasis Type="Italic">Solanaceae</Emphasis>)

Leaves of Solanum virginianum plants were used for protoplast isolation. To support cell wall formation and cell division, protoplasts were cultured in thin alginate layers floated in liquid medium. When protoplasts were plated at a density of 1.0 × 10⁶/ml in Kao and Michyaluk (KMp8) medium supplemented with 0.5 mg/l zeatin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, and 1.0 mg/l α-naphthaleneacetic acid, 42.3% of the dividing cells developed microcalli in 3–4 weeks. Shoot formation via organogenesis of protoplast-derived calli was achieved for 28% of calli transferred to solidified KMp8 medium supplemented with 2.0 g/l zeatin and 0.1 mg/l 3-indol acetic acid in about 2 weeks. Further shoot development was observed in Murashige and Skoog (MS) medium without growth regulators and roots were induced after transfer to MS medium containing 1.0 mg/l 3-indol butyric acid. Regenerated plants have normal morphology.     

Intra- and extra-cellular flux distributions of TCA and glyoxalate cycle and vancomycin production of Amycolatopsis orieantalis grown in different glycerol concentration

The relationship between tricarboxylic acid (TCA) and glyoxalate cycle and the effect of their metabolites levels on the vancomycin production of Amycolatopsis orientalis were investigated in different concentration of glycerol (2.5-20 g/l). Intracellular glycerol levels increased with respect to increases in glycerol concentrations of the growth medium. Extracellular glycerol levels decreased slowly up to 24 h while uptake rates were increased during 36-48 h for 10 and 15 g/l and during 36-60 h at 20 g/l of glycerol. Intracellular citrate, alpha-ketoglutarate, fumarate levels increased up to 10 g/l glycerol concentration. However, intracellular succinate and malate levels were increased up to 15 g/l glycerol. Extracellular citrate, alpha-ketoglutarate, succinate and malate levels increased with respect to increases in glycerol concentration. The highest alpha-ketoglutarate dehydrogenase activity was determined at 15 g/l glycerol. Isocitrate lyase activity showed a positive correlation with the increases in glycerol concentration of the growth medium. Vancomycin production increased with the increases in glycerol concentration from 5 to 10 g/l. These results showed that A. orientalis grown in glycerol containing medium used glyoxalate shunt actively instead of TCA cycle which supports precursors of many amino acid which are effective on the antibiotic production.     

Application of microscopic fungi isolated from polluted industrial areas for polycyclic aromatic hydrocarbons and pentachlorophenol reduction

The growth abilities of fifteen fungal strains isolated fromcontaminated areas, in the presence of xenobiotics compounds mixture (overworked cuttingfluid, crude and waste oil) were examined. Strains with the richest growth were chosen for anthracene, phenanthrene and pentachlorophenol biodegradation in Sabouraudmedium (with initial xenobiotic concentration 250 mg/l in cultures with polycyclicaromatic hydrocarbons and 10 mg/l for the chlorinated substrate). Strains IM 1063and IM 6325 were able to attack phenanthrene forming its derivative 9-phenanthrenolwith the yields 5.22 mg/l and 2.82 mg/l, respectively. Strain IM 1063 and IM 6325 transformed pentachlorophenol to an intermediatecompound – pentachloromethoxybenzene. Final content of pentachloromethoxybenzene reached 3.46 mg/l and3.2 mg/l, respectively. Strain IM 6203 (contrary to other strains) released an intermediateproduct of pentachlorophenol metabolism – 2,3,5,6-tetrachlorohydroquinone(8.73 mg/l substrate remaining and 1.2 mg/l 2,3,5,6-tetrachlorohydroquinone forming).The IM 6203 strain was identified as Mucor ramosissimus. The chlorinatedpesticide degradation by M. ramossimus was improved significantly on a medium with overworked oil. Only 8.3% of pentachlorophenol and 4.3% of 2,3,5,6-tetrachlorohydroquinone in relation to the introduced substrate (10 mg/l) were found, after7 days of incubation. The growth of M. ramosissimus on medium with overworked oil in pentachlorophenol presence was associated with oil emulgation,which enhanced fungal growth and the pesticide degradation.     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

Sugarcane tissue and protoplast culture

Experiments are described which improve the protocols for initiating in vitro cultures of sugarcane and allowing efficient regeneration of plants even after 30 months of callus proliferation. Procedures adopted included use of leaf base explants, CS medium with 3 mg/l 2, 4-D and 0.25 mg/l kinetin for callus initiation and growth, MS medium with 0.5 mg/l IAA and 1 mg/l BAP for shoots, MS medium with 5 mg/l NAA and 7% (wt/vol) sucrose for rooting of shoots. Casein hydrolysate (N-Z amine) significantly shortened the lag period in the growth of sugarcane suspension cultures, but did not increase the rate of growth following the lag phase. Protoplasts isolated from two types of cultures could be grown to re-establish cell cultures but no plants have yet been regenerated derived from isolated protoplasts.     

Production of plantlets from Aegle marmelos nucellar callus

Techniques have been developed for the regeneration of Aegle marmelos from nucellar explants. Slow-growing calli were induced from nucellar explants excised from 90–120 d-old developing fruits. The medium consisted of Murashige and Skoog formulation containing 40 g/l sucrose, 400 mg/l casein hydrolysate, 5 mg/l 1-naphthaleneacetic acid and 1 mg/l kinetin. The basal medium with high concentration (1–5 mg/l) of N6-benzyladenine (BA) and low concentration (0.1 mg/l) of NAA was suitable for regeneration of shoots from 3-month-old calli. Addition of 1 mg/l gibberellic acid (GA3) favoured shoot growth. Callus-derived shoots produced roots and developed into plantlets when transferred to half-strength MS medium supplemented with 0.5 mg/l indole-3-butyric acid (IBA) and 0.5 mg/l NAA. Approximately 5 months were required for the full regenerative process.     

Induction of direct somatic organogenesis in onion (Allium cepa L.) using a two-step flower or ovary culture

A novel method for direct organogenesis in onion (Allium cepa L.) resulting in the formation of multiple shoot structures induced on mature flower buds or ovaries in a two-step culture procedure is described. Flowers were cultured on an induction medium containing 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/l 6-benzylaminopurine (BAP). After 6 days (superior to 3 or 12 days), flowers or extracted ovaries were transferred to a differentiation medium containing 2 mg/l thidiazuron (TDZ). Medium solidification with gellan gum was superior to agar or agar/gellan gum mixture. A similar regeneration frequency was achieved at high (100 g/l) and lower (50 g/l) sucrose content. Regeneration was obtained from all 12 cultivars or inbred lines examined, although the efficiency and the occurrence of hyperhydricity varied depending on genotype and procedure used. Studies of plant growth regulators revealed that in the induction medium, the auxin 2,4-D was superior to 5 mg/l naphthaleneacetic acid or picloram, which partially or completely inhibited regeneration. Omitting cytokinin in the induction medium or substitution of BAP with 2 mg/l 2iP lowered regeneration, while substitution with 1 mg/l TDZ was equally effective. In the differentiation medium, lower concentrations of TDZ (1 and 0.5 mg/l) or substitution of TDZ with 5 mg/l BAP were equally or less effective. Received: 14 October 1998 / Revision received: 19 November 1998 / Accepted: 30 November 1998     

Somatic embryogenesis and plant regeneration from protoplasts of asparagus (Asparagus officinalis L.)

Protoplasts were isolated from embryogenic calli of Asparagus officinalis L. cv. Mary Washington and cultured in 1/2 MS medium with 1 mg/l NAA, 0.5 mg/l zeatin, 1 g/l L-glutamine, 0.6 M glucose and 0.1% Gellan Gum. Protoplasts started to divide after 3–4 d of culture and formed visible colonies after 30 d of culture. The percentage of colony formation (plating efficiency) was 7.2%. The colonies were then transferred onto Gellan Gum-solidified MS medium containing 1 mg/l 2,4-D and 3% sucrose for further growth. Somatic embryos were induced from all colonies of 0.5–1.0 mm size after transferring to 1/2 MS medium lacking growth regulators. After treating these somatic embryos (1–3 mm) in distilled water for a week, 30–40% of them germinated normally and grew into plantlets 20–30 d after transplanting on 1/2 MS medium containing 1 mg/l IBA, 1 mg/l GA₃ and 1% sucrose. These protoplast-derived plants were diploid with 20 chromosomes.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962)