<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from the immature seeds of <Emphasis Type="Italic">Cymbidium faberi</Emphasis>

An in vitro plant regeneration protocol of Cymbidium faberi from immature seeds was established. The immature seeds of 50 days old started to form rhizomes 4 months after they were cultured on hormone free medium. The rhizomes multiplied 5 times when subcultured on the medium containing 1.0 mg l^–1 -naphthalene acetic acid (NAA) for 40 days and more than 90% of the rhizomes initiated shoots within 60 days on the media containing 0.5 or 1.0 mg l^–1 NAA plus 2.0 or 5.0 mg l^–1N⁶-benzylaminopurine (BA). Plantlets were regenerated when the shoots were planted on the basal medium amended with 1 g l^–1 activated charcoal for 50 days and the plantlets grew normally after transplanting.     

Callus induction and plant regeneration of <Emphasis Type="Italic">Spirodela polyrhiza</Emphasis>

Cattle breeding is an important economical activity in Argentina, highly dependent on grass production. In the last decades, grasslands zones were reduced and confined to less productive lands due to the advance of agronomical cultures. Therefore, it is important to develop new strategies to improve forage production. New eco-friendly trends in plant growth promotion include the use of microbial endophytes, but the in vitro studies of plant-bioinoculant interactions is limited by the scarce current technological development. In this work, we use a micropropagation protocol for Lolium multiflorum, developed in a previous work, to study the effect of bacterization with actinobacterial endophytes, isolated from Argentine native grasses, on the growth of L. multiflorum in vitro plantlets. To achieve this objective, L. multiflorum plantlets were inoculated with three Micromonospora strains (SB3, TW2.1 and TW2.2). The results obtained showed that the effect of actinobacterial inoculation depends on the Micromonospora strain used. The inoculation with SB3 promoted plant growth, increasing plant biomass, root length and the rate of plantlets ready to be acclimatized after 4 weeks of in vitro culture. Strain TW2.1 did not show, statistically, differences compared to control treatments, while TW2.2 inhibited plant growth, decreasing plant biomass, root length and the rate of plants ready to acclimatize. Our results showed that Micromonospora strain SB3 could be a good candidate to use in breeding programs for L. multiflorum and other grasses to increase their yield.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.)

Transient expression studies using blueberry leaf explants and monitored by -glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)₃AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 M AS. Explants were then placed on modified WPM supplemented with 1.0 mg l^–1 thidiazuron, 0.5 mg l^–1 -naphthaleneacetic, 10 mg l^–1 kanamycin (Km), and 250 mg l^–1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 E m^–2 s^–1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Tissue culture propagation of Mongolian cherry (<Emphasis Type="Italic">Prunus fruticosa</Emphasis>) and Nanking cherry (<Emphasis Type="Italic">Prunus tomentosa</Emphasis>)

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 M indole-3-butyric acid (IBA) and either 4.44 or 8.88 M 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 M BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 M thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 M) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Feronia limonia</Emphasis> (L.) Swingle from hypocotyl and internodal explants

The hypocotyl and internodal segments from in vitro grown seedlings of Feronia limonia (L.) Swingle (wood apple) were cultivated on Murashige and Skoogs (1962, MS) medium supplemented with N⁶-benzyladenine (BA) or adenine (ADE) or kinetin (KN) at 0.5 to 5 µM. The optimum response was recorded on the medium containing 2 µM BA. An average of 12 and 8 shoots were developed from hypocotyl and internodal explants, respectively, after eight weeks of culture. The shoots were excised, and the residual explants were transferred to fresh medium where again they developed shoots. Up to three such passages resulted in the production of shoots from repeatedly subcultured explants and an average of 24 – 36 shoots per explant was obtained. The in vitro developed shoots produced roots when transferred to half strength MS medium supplemented with 1 µM 1-naphthaleneacetic acid (NAA). The developed plantlets were successfully transferred to mixture of soil, sand and coco-peat (1:1:1) and hardened in controlled environment. Hardened plants were transplanted to soil in greenhouse.     

Shoot multiplication in <Emphasis Type="Italic">Rauvolfia tetraphylla</Emphasis> L. using thidiazuron

The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10M) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5M TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5M indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.     

Cell volume distribution dynamics of <Emphasis Type="Italic">Chlorella</Emphasis><Emphasis Type="Italic">vulgaris</Emphasis> Beij. in batch cultures under continuous light

Cell volume distribution in Chlorella vulgaris cultures coming out of senescence was measured by flow cytometry every 6 h for 114 h in a full-factorial experiment with initial nitrate (420–4200 g NO₃-N l^–1), phosphate (9–186 g PO₄-P l^–1), and continuous light (50–330 E m^–2 s^–1) as treatments. The maxima in median and median absolute deviation (MAD) of cell volume were achieved within 6 h of each other and their timing was not affected by any treatment. Population specific growth rate during the first 66 h calculated from volume distribution changes was significantly affected by light treatment only (p=0.002).Revisions requested 4 November 2004; Revisions received 17 January 2005     

Callus induction and <Emphasis Type="Italic">de novo</Emphasis> regeneration from callus in Guar (<Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>)

Guar (Cyamopsis tetragonoloba L. Taub) is a drought tolerant and multipurpose grain legume cash crop grown primarily under rainfed conditions in several countries. The effect of various growth regulators and their combinations on a variety of explants, namely the embryo, cotyledons, cotyledonary nodes, shoot tip and hypocotyle, has been studied and an efficient system for callus induction and regeneration from callus has been developed. It was established that Murashige and Skoogs culture medium containing 2,4-dichlorophenoxyacetic acid (10.0M) in combination with 6-benzylaminopurine (5.0M) with embryo or cotyledon explants is most suitable for induction of green and friable morphogenic callus, with a range of 82.5–95% of cultured explants responding to callus induction. Efficient de novo shoot regeneration was achieved by culturing the callus obtained on this medium on Murashige and Skoogs medium containing 1-naphthlenacetic acid (13.0M) in combination with 6-benzylaminopurine (5.0M) with a range of 82.1–88.4% of callus clumps producing 20–25 shoots. In vitro rooting of cultured shoots was obtained on half-salt concentration of Murashige and Skoogs culture medium supplied with indole-3-butyric acid (5.0M) on which 82–90% of cultured shoots produced healthy roots. The in vitro regenerated plants were grown to pod setting and subsequent maturity under greenhouse conditions.     

Effects of <Emphasis Type="Italic">in vitro</Emphasis>rooting environments and irradiance on growth and photosynthesis of strawberry plantlets during acclimatization

Strawberry (Fragaria ananassaDuch. cv. Fengxiang) plantlets were cultured under two in vitroenvironments for rooting, and then acclimatized under two ex vitroirradiance conditions. At the end of rooting stage plant height, fresh weight and specific leaf area of T1-plants grown under high sucrose concentration (3 sucrose), low photosynthetic photon flux density (30 mol m^–2 s^–1) and normal CO₂ concentration (350–400 l l^–1) were significantly higher than those of T2-plantlets grown under low sucrose concentration (0.5), high photosynthetic photon flux density (90 mol m^–2 s^–1) and elevated CO₂ concentration (700–800 l l^–1). But T2-plantlets had higher net photosynthetic rate (Pn), effective photochemical quantum yield of PSII (_PSII), effective photosynthetic electron transport rate (ETR), photochemical quenching (q_P) and ratio of chlorophyll fluorescence yield decrease (Rfd). After transfer, higher irradiance obviously promoted the growth of plantlets and was beneficial for the development of photosynthetic functions during acclimatization. T2-plantlets had higher fresh weight, leaf area, _PSII and ETR under higher ex vitroirradiance condition.     

Strategic timing of nitrogen fertilization to increase root biomass and nitrogen-use efficiency of <Emphasis Type="Italic">Lolium perenne</Emphasis> L.

Aims

During the first days after harvest of Lolium perenne L., N remobilized from roots and stubble forms the main N source for regrowth. Low N uptake from the soil during this period may lead to N loss if N fertilizer is applied too soon. Furthermore, temporary N deprivation has been found to stimulate root growth. We therefore hypothesized that a strategic delay in N application after harvest may improve N-use efficiency of L. perenne grassland by increasing root biomass and reducing N loss.

Methods

In a laboratory and field experiment with L. perenne, we delayed N fertilizer application after harvest for 0, 3, 6, 9 and 12 days, repeated this for up to six harvest cycles, and determined effects on herbage yield, herbage N uptake and root biomass.

Results

In both experiments, delaying N application for up to 12 days had no significant effect on root biomass or total herbage N uptake, but it significantly reduced total herbage yield in the laboratory experiment. Total yield tended to be highest when N application was delayed for 3 days. Two growth periods in the field experiment showed significantly higher N uptake when N application was delayed, possibly due to rainfall-induced N losses in the treatments with shorter delay.

Conclusions

Our results do not provide evidence that delaying N application improves N-use efficiency of L. perenne grassland by increasing root biomass. However, strategic timing of N fertilizer application based on rainfall forecasts could contribute to improve N-use efficiency by reducing N losses from leaching and denitrification.

     

<Emphasis Type="Italic">Sarcocystis</Emphasis> sp. in red foxes (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) from northern Croatia

The prevalence of Sarcocystis sp. was assessed in foxes from northern Croatia (city of Varadin). The intestine of 50 (29 male and 21 female) foxes aged 1–5 years, killed during the rabies control campaign, were examined. Sarcocystis sp. was identified in 62% of the fox intestinal samples examined. There was no sex difference in the rate of invasion. However, the rate of invasion differed significantly between the animals aged 1 year (47%), and those aged 2 (71%) and 3–5 years (71%).     

Bioavailability of zinc in the sediment to the estuarine amphipod <Emphasis Type="Italic">Grandidierella</Emphasis><Emphasis Type="Italic">japonica</Emphasis>

The utility of interstitial water concentrations of metals and simultaneously extracted metals/acid-volatile sulfide differences (SEM–AVS) in two seasons were investigated to explain the biological availability of zinc in sediments to benthic organisms exposed in the laboratory. The amphipod Grandidierella japonica was exposed, in 10-day acute toxicity tests, to clean sediment spiked with zinc to obtain nominal treatments ranging from 0.25 to 74.4 mol g^–1 dry weight with respect to the molar difference between SEM_Zn and AVS. When the molar difference between SEM_Zn and AVS (i.e., SEM–AVS) was <0 mol g^–1, the concentration of zinc in the sediment interstitial water was low and few adverse effects were observed for any of the biological endpoints measured. Conversely, when SEM_Zn–AVS exceeded 0 mol g^–1, the concentration of zinc in the interstitial water and amphipod mortality increased. These data compare favorably with observations made in short-term exposures and thus support the use of AVS as a normalization phase for predicting toxicity in metal-contaminated sediments in different season.     

Selection of atrazine-resistant plants by <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

The effects of atrazine on cotyledon cultures of Capsicum annuum (L.) cv. G₄ were investigated with a view of establishing a system for in vitro selection of resistant mutants. At low levels of herbicide produced little growth inhibition, some chlorophyll loss occurred associated with the production of albino shoots. At 20 mg l⁻¹ atrazine bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenized cotyledon explants resulted in production of herbicide-resistant plants on medium containing selective levels of sucrose (0.5%) and atrazine (20 mg l⁻¹). Differential morphogenetic responses were observed when the levels of sucrose (0.5–5%) were altered. Shoot regeneration was maximum in 2 sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3%–5%). However, lowering of sucrose concentration from 2 to 0.5% caused complete bleaching of explants and permitted the selection of herbicide-resistant plants. Complete atrazine-resistant plantlets were obtained after rooting of regenerated green shoots on rooting medium containing 10 mg l⁻¹atrazine, 1.0 mg l⁻¹IAA and 0.5% sucrose. Leaf-segment assay of differentiated plants revealed that all regenerants were resistant to the atrazine. Reciprocal crosses between atrazine-resistant and -sensitive plants showed a non-Mendelian transmission of resistance trait.     

Purification and characterization of an<Emphasis Type="Italic"> N</Emphasis>-acetylglucosaminidase produced by a<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> strain which controls<Emphasis Type="Italic"> Crinipellis perniciosa</Emphasis>

Isolate 1051 of Trichoderma harzianum, a mycoparasitic fungus, was found to impair development of the phytopathogen, Crinipellis perniciosa, in the field. This Trichoderma strain growing in liquid medium containing chitin produced substantial amounts of chitinases. The N-acetylglucosaminidase present in the culture-supernatant was purified to homogeneity by gel filtration and hydrophobic interaction chromatography, as demonstrated by SDS-PAGE analysis. The enzyme had a molecular mass of 36 kDa and hydrolyzed the synthetic substrate -nitrophenyl-N-acetylglucosaminide (NGlcNAc) with Michaelis–Menten kinetics. Maximal activities were determined at pH 4.0 and a temperature range of 50–60°C. K _m and V _max values for NGlcNAc hydrolysis were 8.06 moles ml^–1 and 3.36 moles ml^–1 min^–1, respectively, at pH 6.0 and 37°C. The enzyme was very sensitive to Fe³⁺, Mn²⁺ and Co²⁺ ions, but less sensitive to Zn²⁺, Al³⁺, Cu²⁺ and Ca²⁺. Glucose at a final concentration of 1 mM inhibited 65% of the original activity of the purified enzyme. Determination of the product (reducing sugar) of hydrolysis of C. perniciosa mycelium and scanning electron microscopic analysis revealed that the N-acetylglucosaminidase hydrolyses the C. perniciosa cell wall.     

Studying allozyme variation in sexual and apomictic <Emphasis Type="Italic">Taraxacum</Emphasis> and <Emphasis Type="Italic">Pilosella</Emphasis> (Asteraceae) populations

Allozyme spectra of peroxidase, esterase, superoxid dismutase, tyrosinase, alcohol dehydrogenase, lactate dehydrogenase, and acid phosphatase were examined in populations of sexual (Taraxacum serotinum and Pilosella echioides) and apomictic (T. officinale and P. officinarum) plant species. The heterozygosity in these populations (0.455–0.620) proved to be considerably higher than the average level characteristic of plant populations (0.058–0.185). The populations examined did not differ in the mean phenotype number , i.e., they exhibited the same diversity (3.188–3.380). The proportion of rare phenotypes h also did not differ between the sexual and apomictic species of the same genus, whereas this parameter in the Pilosella populations (0.150–0.174) was significantly higher than in the Taraxacum ones (0.093–0.114). The populations were characterized by numerous isozyme spectra (more than 11 per populations) and displayed multiple allelism (the mean allele frequency was 3.63–4.38 per locus). They exhibited a high percentage of rare (occurring at a frequency lower than 5%) spectra (35–80%). This indicates that agamic complexes, to which these populations belong, may have a more complicated genetic structure of both apomictic and sexual populations than the species that do not belong to agamic complexes.Translated from Genetika, Vol. 41, No. 2, 2005, pp. 203–215.Original Russian Text Copyright © 2005 by Kashin, Anfalov, Demochko.     

Efficient regeneration of <Emphasis Type="Italic">Brassica napus</Emphasis> L. hypocotyls and genetic transformation by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

An efficient system for shoot regeneration and Agrobacterium-mediated gene transfer into Brassica napus was developed through the modification of the culture conditions. Different concentrations of benzyladenine (1.5, 3.0 and 4.5 mg dm^–3) and thidiazuron (0.0, 0.15 and 0.30 mg dm^–3) were evaluated for shoot regeneration of 7, 14 and 21-d-old hypocotyl explants. Maximum shoot regeneration frequency was obtained in 21-d-old explants using 4.5 mg dm^–3 benzyladenine and 0.3 mg dm^–3 thidiazuron. Under above culture condition, the highest percentage of shoot regeneration frequency was 200 %. Agrobacterium-infected explants grown on the selection medium gave rise to transgenic shoots at a frequency of 11.8 %. Transformed shoots rooted when cultured on a medium supplemented with 2 mg dm^–3 of indolebutyric acid and 10 mg dm^–3 kanamycin. The rooted plantlets were successfully established in the soil and developed fertile flowers and viable seeds. Evidences for transformation were confirmed by GUS assay and PCR analysis.     

Synteny between a major heading-date QTL in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) and the<Emphasis Type="Italic"> Hd3</Emphasis> heading-date locus in rice

The genetic control of induction to flowering has been studied extensively in both model and crop species because of its fundamental biological and economic significance. An ultimate aim of many of these studies has been the application of the understanding of control of flowering that can be gained from the study of model species, to the improvement of crop species. The present study identifies a region of genetic synteny between rice and Lolium perenne, which contains the Hd3 heading-date QTL in rice and a major QTL, accounting for up to 70% of the variance associated with heading date in L. perenne. The identification of synteny between rice and L. perenne in this region demonstrates the direct applicability of the rice genome to the understanding of biological processes in other species. Specifically, this syntenic relationship will greatly facilitate the genetic dissection of aspects of heading-date induction by enabling the magnitude of the genetic component of the heading-date QTL in L. perenne to be combined with the sequencing and annotation information from the rice genome.Communicated by Q. Zhang     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation and development of herbicide-resistant sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> species hybrids) using axillary buds

Direct regeneration from explants without an intervening callus phase has several advantages, including production of true type progenies. Axillary bud explants from 6-month-old sugarcane cultivars Co92061 and Co671 were co-cultivated with Agrobacterium strains LBA4404 and EHA105 that harboured a binary vector pGA492 carrying neomycin phosphotransferase II, phosphinothricin acetyltransferase (bar) and an intron containing -glucuronidase (gus-intron) genes in the T-DNA region. A comparison of kanamycin, geneticin and phosphinothricin (PPT) selection showed that PPT (5.0 mg l^–1) was the most effective selection agent for axillary bud transformation. Repeated proliferation of shoots in the selection medium eliminated chimeric transformants. Transgenic plants were generated in three different steps: (1) production of putative primary transgenic shoots in Murashige-Skoog (MS) liquid medium with 3.0 mg l^–1 6-benzyladenine (BA) and 5.0 mg l^–1 PPT, (2) production of secondary transgenic shoots from the primary transgenic shoots by growing them in MS liquid medium with 2.0 mg l^–1 BA, 1.0 mg l^–1 kinetin (Kin), 0.5 mg l^–1 -napthaleneacetic acid (NAA) and 5.0 mg l^–1 PPT for 3 weeks, followed by five more cycles of shoot proliferation and selection under same conditions, and (3) rooting of transgenic shoots on half-strength MS liquid medium with 0.5 mg l^–1 NAA and 5.0 mg l^–1 PPT. About 90% of the regenerated shoots rooted and 80% of them survived during acclimatisation in greenhouse. Transformation was confirmed by a histochemical -glucuronidase (GUS) assay and PCR amplification of the bar gene. Southern blot analysis indicated integration of the bar gene in two genomic locations in the majority of transformants. Transformation efficiency was influenced by the co-cultivation period, addition of the phenolic compound acetosyringone and the Agrobacterium strain. A 3-day co-cultivation with 50 M acetosyringone considerably increased the transformation efficiency. Agrobacterium strain EHA105 was more effective, producing twice the number of transgenic shoots than strain LBA4404 in both Co92061 and Co671 cultivars. Depending on the variety, 50–60% of the transgenic plants sprayed with BASTA (60 g l^–1 glufosinate) grew without any herbicide damage under greenhouse conditions. These results show that, with this protocol, generation and multiplication of transgenic shoots can be achieved in about 5 months with transformation efficiencies as high as 50%.Abbreviations BA 6-Benzyladenine - CaMV Cauliflower mosaic virus - GUS -Glucuronidase - Kin Kinetin - NAA -Naphthaleneacetic acid - Nos Nopaline synthase - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - PPT Phosphinothricin - YEP Yeast extract and peptone