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Production of transgenic carnation with antisense ACS (1-aminocyclopropane44-carboxy late synthase) gene
104-107.Views:175Dianthus chinensis and Dianthus caryophyllus varieties were tested for shoot regeneration from leaf and petal explants and transformed with Agrobacterium tuniefaciens strains (EHA 105 and LBA 4404) harbouring an apple derived ACS cDNA in antisense orientation in order to reduce ethylene production and influence the ethylene dependant traits in carnation. After transformation regenerating shoots were selected on MS medium containing 50-75-100-125-150 mg/1 kanamycin and supplemented with 1 mg/1 BA, 0.2 mg/1 NAA. Transgene integration was proved by PCR analysis with npt II spcific primers followed by Southern hybridisation of DNA isolated from green shoots on medium containing 150 mg/1 kanamycin. Several putative transformants were subjected to RT-PCR in order to examine the npt 11 expression at mRNA level. Both the transformant and the non-transformant plants were potted into glasshouse to observe the effect of changed ethylene production on flowering time, petal senescence and vase life.
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The effect of different carbohydrates on the multiplication of Hosta cultivars
115-117.Views:129The effect of seven concentrations of two carbohydrate sources were compared to determine the best source and the most suitable source and concentration for micropropagation of some Hosta cultivars: H. 'Gold Haze', H. 'Gold Drop' and H. 'Dew Drop'. 0, 5, 10, 20, 30, 40 and 50 g/1 sucrose or glucose were added to a MS basic medium supplemented with 3 mg/1 kinetin and 0.1 mg/1 IAA. For 'Gold Haze' 40 g/1 sucrose proved to be the best source and concentration, the proliferation ratio was 15 shoots per explant. Thirty g/1 sucrose concentration was the optimum for 'Gold Drop', the proliferation rate was 14.6 shoots per explant. In 'Dew Drop,' the best results were obtained with 30 g/1 sucrose but 40 g/l sucrose gave good results too. Both cultivars rooted well on these media. On glucose containing media, very low propagation rates were found in all concentrations and all examined cultivars.
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Morphological, physiological features and differences of Vriesea splendens ’Fire’ plants during in vitro multiplication and rooting
Views:119During in vitro multiplication and rooting of Vriesea splendens ’Fire’, 0.1, 0.2, 0.4 and 0.8 mg l-1 benzyladenine (BAP), benzyladenine-riboside (BAPR), kinetin (KIN), meta-topoline (MT), indole-butyric acid (IBA) and naphthalene-acetic acid (NAA) were added to basal Murashige and Skoog (1962) MS medium. As compared to the hormone-free control, plants developed significantly more shoots on medium supplemented with almost all cytokinins (excepting KIN), especially BAP resulted the highest multiplication up to almost 26 shoots. Enhancement of cytokinin concentrations increased shoot number (and in case of BAP, peroxidase activity) but decreased plant height and rooting parameters. Regarding root production, both auxins were definitely beneficial (0.2 mg l-1 NAA resulted more than 7.5 roots and higher auxin concentrations efficiently stimulate root elongation); however, KIN had similar effects. After a three-month duration time of acclimatization, we observed that plants which were previously cultured on medium containing certain cytokinins (KIN in all doses and 0.1 mg l-1 MT) or both auxins had greater survival, moreover, as negative after-effect, higher cytokinin concentrations reduced the number of survived specimens.
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Fire blight (Erwinia amylovora) susceptibility of old Hungarian apple cultivars
35-38.Views:152The aim of the Hungarian apple breeding program started in the Department of Fruit Science was to find resistant apple cultivars against major diseases (scab, powdery mildew, fire blight). The outbreak of fire blight (Erwinia amylovora) in 1996 motivated us to search new resistant sources principally from old traditional apple cultivars. First of all, cultivars have been gathered since 1997 from Carpathia (Visk) and evaluated between 2001 and 2003. In this recent study evaluation of resistance of old Hungarian genotypes to fire blight collected from the English National Fruit Collection (Brogdale, Faversham) is presented.
13 old Hungarian apple cultivars in 2002, and 38 genotypes in 2005 have been evaluated. We used `Idared' and 'Jonathan M41' as susceptible controls and 'Liberty' and 'Remo' as resistant ones. Shoots of two-year-old potted plants were inoculated with a mixture of virulent E. amylovora isolates (Ea2, Ea60, Ea67) at a concentration of 5 x 108 cells/ml. Resistance of apple cultivars was evaluated weekly, four times after inoculation by disease severity of symptoms. Numbers of bacterial colonies in 1 cm length shoot were determined in the fourth week after infection.
8 cultivars in 2002 and 9 cultivars in 2005 displayed notable resistance to fire blight based on one-year data. Based on the coincident data of both years, out of the cultivars collected also from Carpathia ‘Pónyik', and `Sikulai' were found to be resistant and gene sources additional old Hungarian valuable apple genotypes could be selected: `Szabadkai szercsika' and `Tordai piros !davit'. The cultivar `Szemes alma' originated from Visk has proved to be recurrently resistant.
The number of bacterial cells in shoots of the investigated cultivars correlated with the severity of symptoms. With this method, which was introduced by us earlier, we can screen cultivars displaying weak visible symptoms, which cannot be proposed as a source of resistance because of their latent infection.
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Inhibition and recovery of germination and growing ability of seedlings under and after osmotic stress induced by polyethylene glycol in 8 pea genotypes
53-59.Views:130Germination and early seedling growth of eight pea genotypes were examined under and soon after different (5%, 10%, 15%, and 20%) PEG treatments. Seeds were germinated on PEG solution for 3 or 6 days and then further germinated and cultured on filter paper moistened by water for 3 and 6 days. The length and fresh weight of shoots and roots of seedlings were measured and used for evaluation of genotypes. Roots were less inhibited by osmotic stress than shoots similarly to other plant species. The variability among the genotypes was the greatest in the case of shoot growth at 5% PEG treatment and in the case of root growth at 15% PEG treatment. Results suggest that growing responses of genotypes after cessation of stress are more suitable for the evaluation of their osmotic tolerance, than their responses expressed during in vitro stress conditions. Genotypes with relatively high or low osmotic stress tolerance, respectively, could be distinguished with 6 days after recovery from 3-day-long 15% PEG treatment concerning the rate of shoot weight to root weight.
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In vitro regeneration from cotyledons of watermelon
96-98.Views:166Cotyledonary segments of five different genotypes of watermelon were used to induce organogenesis. Five different hormone combinations were applied to enhance the induction of shoot formation on the surface of the segments. The phases of organogenesis were followed with light and scanning electron microscope. Shoots were obtained after four weeks, then the shoots were transferred to hormone free medium for root induction.
This method of regeneration can be applied in transformation experiments. GUS histochemical assay was made to check the expected success of using Agrobacterium for the transformation.
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Relaco: a product to reduce water shoot formation around pruning wounds in pome fruit trees
30-31.Views:108Larger cuts made during the winter pruning of pome fruit orchards a number of serious problems: the wounds serve as entrances for several branch-infecting pathogens, the numerous water shoots formed around the cut edges consume assimilates, shade the developing fruits, make the canopy dense, reduce the effectiveness of the pest management and cause extra work during next seasons pruning work. These problems can be eliminated by using proper wound protectants, like Relaco, a new Romanian wound protectant to cover larger pruning cuts of apple, pear and quince trees.
The treaments by Relaco resulted in a significant decrease in both the number and total length of water shoots formed around the cut egdes.
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Influence of aromatic cytokinins on shoot multiplication and their after-effects on rooting of apple cv. Húsvéti rozmaring
84-87.Views:219Different aromatic cytokinins (BA, BAR, TOP and KIN) were tested alone or in combination for the shoot proliferation response of ‘Húsvéti rozmaring' apple scion. The best multiplication rate was achieved by dual cytokinin application (1 mg 1-1 BA + 1.5 mg 1-1 KIN). The rooting capacity was affected considerably by the position of shoots: transfer of the three-week-old shoots to the same or other proliferation medium in vertical position inhibited the following rooting totally. Post-effects of different cytokinins (BA and TOP) on subsequent rooting could be detected: BA increased the number of roots markedly, while TOP resulted in significantly longer roots.
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Yielding capacity and regeneration of different ages fruit-bearing shoots in sour cherry
55-57.Views:180The yielding potential and regeneration (i.e. germination of hidden buds) after pruning of the fruit-bearing shoots of different ages were compared in sour cherry cultivars pruned to spindle shape. The examinations were carried out in order to determine the optimal pruning strategy resulting in the highest long-term yielding of sour cherry cultivars with intensive crown shape (free and thin spindle shape). According to our results, there are great differences among the studied self-fertile sour cherry cultivars in yielding and regeneration, which are worthwhile to be considered in determining the method and degree of pruning.
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Obtention of new ornamental leaf variants of giant reed (Arundo donax L.) originated from somatic embryogenesis and their photosynthetic parameters
18-24.Views:441Giant reed (Arundo donax L.) is a perennial rhizomatous herbaceous plant, it has been widespread all over the world by human activities. It is a 2-8 meter high, polyploid, sterile species (not produce viable seeds), it can be propagated only by vegetative methods (rhizomes, stem cuttings, in vitro biotechnological methods). It has considered promising dedicated energy crop thanks to high biomass production (20-40 dry tons per hectare depending on microclimate), adaptability of different kind of soils and environment and low energy input required for its cultivation. It has been utilized for energetic purposes, biogas/bioethanol production, cellulose/paper production and ornamental purposes. The objectives of the present study were to determine morphological properties of new ornamental giant reed leaf variants originated from somatic embryogenesis and to evaluate their photosynthetic pigment content and photosynthetic activities. The most typical changes was the appearance of different colour (white, yellow, light green) longitudinal stripes on leaves (also on petiole and on leaf blade). It was significant differences (p<0.05) between green and leaf variants in case of photosynthetic pigments content and photosynthetic activity (Fv/Fm value). There was no detectable chlorophyll a or b content in the white bands of leaves and albino shoots. Total chlorophyll content of the white striped leaf variety was more than twice than the light green leaves. Photosynthetic activity and content of photosynthetic pigments also confirm and determine the morphological characteristics and growth habit of leaf variants.
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New in vitro micrografting method for apple by sticking
79-83.Views:262The requirements for in vitro micrografting in apple are described. In vitro multiplicated shoots of cv. Royal Gala were the sources of rootstocks and scions after different pre-treatment, respectively. Oxidative browning of cut surfaces could be inhibited by the use of antioxidant mixture during grafting process. Scion base cut in v-shape was stuck by 1% agar-agar solution into the vertical slit of rootstock. There was no any displacement and the rate of fused and further developed grafts was 95 percent. Agar-agar between the rootstock and scion made the transport of different materials possible and hold the graft units together until the fusion took place. Fusion was proved also by histological studies. Some of in vitro micrografts were planted and acclimatisated and the survival was 100 percent.
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Effect of Erwinia amylovora infection on peroxidase enzyme activity in resistant apple cultivars
37-39.Views:155Two apple cultivars that display enhanced resistance to fire blight (causal agent: Erwinia amylovora) were selected. The aim of the present study was to characterize the peroxidase (POD) enzyme activity of `Szemes alma' (a historical cultivar) and MR-03, (a Hungarian multiresistant hybrid of 'Prima') and compare them to susceptible 'Jonathan M 40' and resistant 'Remo' controls. Peroxides enzyme activity during E. amylovora infections was investigated in artificially infected apple shoots. Increases in enzyme activities were observed in a `Jonathan M40' and in 'Remo', MR-03, `Szemes alma' cultivars. There was a consistent relationship between total enzyme activity and fire blight disease severity. High activity of the peroxidase was positively correlated with the degree of resistance to fire blight. A general hypothesis that POD activity is related to fire blight susceptibility/resistance is supported by our results.
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In vitro multiplication and hardening of grapevine plants in aeriated media
15-18.Views:212In vitro cultures have widely been used in horticulture for rapid multiplication of new varieties and clones as well as to produce pathogen-free stock material. To improve efficient hardening and transfer in vitro grown grapevine plants were multiplied by cutting them into single-node internodes with the whole leaf. Microcuttings including the shoot tips were rooted in granulated perlite moisted with tapwater under sterile conditions. After 2-3 weeks the rooted microcuttings were supplied by nutrients and hardened by gradual opening and finally by complete removal of the lids of jars or plastic boxes used for growth. Using this method microcuttings of Vitis vinifera cvs. „Chardonnay", „Cabernet franc", „Riesling" and „Sauvignon blanc" and the rootstock varieties Vitis riparia x Vitis cinerea cv. „Barrier" and Vitis berlandieri x Vitis rupestris cv. „Richter 110" formed new roots and shoots and 100% of the tested plants survived the acclimatization procedure. Similar results were obtained when perlite was replaced with rockwool-, or pit-pot blocks. This method may highly increase the efficiency of producing pathogen-free propagating material and new transgenic lines.
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Fruit drying traditions in Hungary
7-10.Views:380Since the early eras of human history, the gathered products (roots, shoots, crops, fruits etc.), the environmental conditions of the preservation of the animals caught and the fishes; and accidental observations have been born new methods. Under cold and moderate climates, the effect of permanent frost, the use of ice as a means of curing as in dry, desert areas is the drying or exsiccation and the dehydration of salt. For convenience reasons, some of these methods have been forgotten, but the resurfacing of different fruits seems to resume. The short review gives an overview of the methods of drying used in the Carpathian Basin, especially among the Hungarian ethnic groups, which by the Hungarian ethnology thoroughly explored. The author attaches outstanding importance to the fruit-drying industry, because dried-ups at industrial scale are not equivalent to traditional low-fat lime scales. Excellent drying products are much better in terms of eating habits, with modern methods and small-scale production relatively easy to produce.
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A study of processes active in regeneration of different sweet cherry varieties
55-57.Views:217Cherries belong to the group of fruit trees, which require little attention in pruning. The process of getting bald branches is less accentuated, which is associated with the light requirement of organs changing along with the aging process. The growing habits of the varieties (length and number of growing shoots, etc.) is closely related with the productivity of fruiting parts. The ability of regeneration and the functional effectiveness of fruiting structures are expressed by the distribution according to their age. The differences registered justify the necessity to develop pruning policies for each particular type of variety. For that purpose, we have to examine the age dependency of the processes related to regeneration, which may serve as starting point of a pruning strategy. Growth of some parts should be stimulated in order to develop or regenerate fruiting parts. Some branches need to be rejuvenated, partially, as an antidote to progressive senescence. Every intervention is aimed to strengthen the development of the most productive constructions. Reactions induced by rejuvenation should be expressed in fruiting intensity. A pruning program adapted to the particular variety as well as to the actual phase of a rotating pruning system should anticipate results.
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Reactions of different plant organs of pear cultivars to Erwinia amylovora infection
17-21.Views:137Research project has been initiated in 1999 with the aim of evaluating the degree of susceptibility/resistance of pear cultivars grown in Hungary to fire blight disease caused by Erwinia amylovora. The recently selected promising cultivars were also examined. Inoculation experiments were conducted in controlled greenhouse conditions because of quarantine regulations in Hungary. Following the disease process, development of symptoms of plant organs (shoots, flower parts, fruits) was observed. Suspension of two E. amylovora strains (Ea 21, Ea 23) isolated from pear was used in a mixture (5x108 cells x m1-1) for the inoculation. Twenty-six pear cultivars were examined and grouped into four categories: low susceptibility, moderately susceptible, susceptible and very susceptible. Most of the cultivars were susceptible or very susceptible while some promising 'Eldorado', 'Harrow Delight' and `Hosui' showed low susceptibility.
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Single and dual effects of different cytokinins on shoot multiplication of different apple scions
76-78.Views:178Shoot multiplication responses of three apple scions to different concentrations of BA and BAR as single source of cytokinins and in combination with two concentrations of KIN were studied. The effects of hormones depended on genotype, type and interactions of different cytokinins. Use of BAR significantly enhanced the shoot multiplication of cv. Jonagold (6.5 shoots per explant). The multiplication rate of cv. Jonagold could not be improved by using the combination of BAR and KIN. The best proliferation was achieved by 1.0 mg 1-1 BA combined with 1.0 mg 1-1 KIN of cv. Prima..(8.1) and of cv. Galaxy (10.4).The effect of 0.5 mg 1-1 BA along with 1.5 mg 1-1 KIN was similar on multiplication rate (10.9) of cv. Galaxy.
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Preliminary results of renewal pruning an 18 years old sour cherry trees
41-42.Views:172Sour cherry is a light demanding fruit species. As most of the crop is developing from buds on one year old shoots and 2-yearold wood, the formation of bare wood can be seen apparently in a badly maintained canopy. The formation of „whip shoots” indicates the degree of bare wood. Unfortunately in many gardens and commercial orchards sour cherry trees resemble willow trees. Owners often decide to grub old orchards, although the useful cropping lifespan of the trees planted in a correct spacing can be 20-30 years on a move vigorous rootstock. Therefore renewal pruning can contribute to the full renewal of the orchard, but the length of its effect is depending on the growth characteristics and renewal capabilities of the cultivar. The growth characteristics of the cultivars are different. The regeneration capabilities of different aged wood are also different. These differences emphasize the need for cultivar specifi c pruning. Knowing the reactions to renewal pruning, we can ensure regular high crops with excellent quality by applying cultivar specifi c rotation pruning.
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First selections of the Hungarian apple breeding program for multiple resistance
9-13.Views:149The aim of the first Hungarian apple breeding program for multiple resistance started in the beginning of the nineties is to widen Hungarian apple assortment by good quality, resistant apple cultivars with excellent productivity and ecological capability to the most important fruit growing areas in Hungary. In the first years of seedling production we made early selection for susceptibility to apple scab in greenhouse. Alter this, field observation of susceptibility to powdery mildew, scab and canker and a yearly negative selection was carried on. From 1997, fruit quality was evaluated as well, and from 2001 the resistance of shoots to Erwinia ainylovora (Burrill/Winslow et al.) was examined using inoculations in greenhouse conditions. From the progenies of crosses in 1992 and 1993, six candidates were announced to national recognition out of hybrids examined for more than a decade. Descriptions of these selections from 'Prima' progenies and the most important data of their resistance, growing habit, morphological characteristics and fruit quality are shown in this article.
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Model experiments for establishment of in vitro culture by micrografting in apple
47-49.Views:208Micrografting was used in our experiments for establishment of in vitro culture from one rootstock (`JTE-F') and three scion cultivars (`Remo', 'Rewena' and `Reanda') of apple. Shoot tips of these cultivars were harvested from field and grafted onto in vitro rootstock cultivars. Their survival and development were studied. 42-93% of shoot tips survived and developed further depending on cultivar. Impermanent browning of sticking agar-agar could be observed in 21-25% of the micrografts depending on cultivars but discolouration of agar-agar ceased within one week and did not cause any death of shoot tips. We used micrografting successfully for establishment of in vitro culture from cultivars, from which earlier with conventional methods the culture establishment was not possible because of hard tissue browning. However, further studies are necessary to ensure the survival and development of shoots after removing them from micrografts.
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Investigation of flowering dynamics of the basil (Ocimum basilicum L.) and its consequences in production
74-80.Views:176The flowering dynamics of Ocimum basilicum L., (a common population maintained at the UHFI) was studied with the aim to create an exact and practically applicable method for definition of phenological phases.
According to our observation the development of individual flowers of the basil can be characterized by 8 distinguishable phases, which must be considered for description of the actual phenological stage of a spike.
An accurate model was created for the unambiguous description of flowering process of different flowers within the spike and for the individual plant as a whole. The new flowering index formula is calculated from the number of flowers weighed by their phenological phases.
The time dependence of flowering is presented by functions fitted to values of the flowering index. The results reflected different patterns of the main inflorescence, the inflorescences formed on the side shoots in the first or in the second half of the flowering period. However, for description of the flowering process of the whole plant, a sigmoid function proved to be the appropriate model.
The accumulation process of the essential oil could be characterized by the flowering index values. They showed close correlation (r=0.964) at high probability.
The new method assures an exact definition of phenological phases in basil. Its application seems to be optimal during breeding procedures, seed production and even in production of high quality drugs, according to the requirements of the Good Agricultural Practice (Guidelines for GAP, 1998).
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Effect of reduced spray programmes on incidences of apple scab, powdery mildew and codling moth damage in environmentally freindly apple production systems
75-78.Views:700Still a large amount of pesticides and spary applications are used in environmentally friendly fruit production systems; therefore, the aim of our study was first to test the in vitro effeicacy of some fungicides against a key apple disease (apple scab), and secondly to evaulate the effectiveness of reduced spray programmes against apple scab, powdery mildew and coding moth in integrated and organic apple orchards. In vitro efficacy of 7 fungicides (Champion 50 WP, Kocide 2000, Nordox 75 WG, Olajos rézkén, Kumulus S, Rézkén, Rézoxiklorid) and another 6 fungicides (Score 25 EC, Efuzin 500 SC, Systane, Folicur Solo, Zato Plusz, Rovral) approved in organic and integrated production systems, respectively, were tested against apple scab. Altogether four spray programmes were performed i) standard integrated: sprays followed by forecasting systems during the season, ii) reduced integrated: sprays followed by forecasting systems but only 75% of the spray numbers used during the season-long spray programme, iii) standard oragnic: sprays applied every 7–14 days during the season and iv) reduced organic: 60% of the spray numbers used during the season-long spray programme. In vitro results showed that fungicides (with active ingredients of copper and sulphur) applied in organic production showed relatively high percent growth capacity of the apple scab fungus. Rézkén showed the highest and Kumilus S the lowest efficacy against apple scab. Fungicides applied in integrated production showed relatively low percent growth capacity of the apple scab fungus. Score 25 EC showed the highest and Rovral the lowest efficacy against apple scab. Field study showed that reduced spray programmes did not increase significantly scab incidence in the integrated field. However, scab incidence increased significanly (above 30%) in the reduced spray programme for the organic orchard.Mildew incidence was low (below 5%) in both integrated and organic spray programmes. Mildew incidence on both shoots and fruits increased significanly in the reduced spray programme for the organic orchard. Incidence of codling moth damage was affected the most by standard vs. reduced spray programmes. Though incidence remained below 10% in the integrated plots, the incidence was significantly higher in the reduced spray programme compared to the standard programme. Similar results were obtained for organic spary programmes, but the incidence was 10 times higher and the differences among the two programmes were larger.,
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Effects of indole-3-butyric acid levels and activated charcoal on rooting of in vitro shoots of apple rootstocks
25-28.Views:277Rooting responses of rootstocks cvs. JTE-F1, M. 26 and MM. 106 were studied to different concentration of IBA in root induction media and to presence of activated charcoal in root elongation media. High rooting rate (>90%) could be achieved in cvs. JTE-H and M. 26, while cv. MM. 106 showed weak rooting ability at each IBA level tested. Increasing IBA content depressed the rooting only in cv. M. 26. Presence of activated charcoal decreased considerable the rooting rate in cv. M. 26 and decreased the number of roots in cvs. JTE-H and M. 26. These cultivars developed longer roots on media containing activated charcoal, while cv. MM. 106 did not showed any reaction for it.
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Real-case application of mycorrhizal inoculums on Capsicum annuum L. var. longum cv. Szegedi and Kalocsai
75-79.Views:187The aim of this study was to test the use of commercially available arbuscular mycorrhiza (AM) inoculant Symbivit, a mixture of six species of Glomus spp., in spice pepper field cultivation. The inoculants containing arbuscular mycorrhizal fungi (AMF) was able to establish a symbiosis in the rhizosphere of pepper plants and mycorrhizal inoculation increased fresh and dry weights of shoots of spice pepper cv. Szegedi and only fresh weight of Kalocsai type. There were no significant differences in the root weights due to treatment only in fresh weight of Kalocsai pepper type. Treated plants of both variants exhibited an increase in cumulative crop production compared with control non-treated plants and the growth response of pepper was higher for var. Szegedi than var. Kalocsai. Mycorrhizal inoculation had a great positive effect on external hyphal length of AMF also showing differences in that between Kalocsai and Szegedi variants. The root colonization showed seasonality by treated and non-treated plants. The lowest degree of colonization was observed in June in general and colonization percent increased during vegetative development and there was a slight decrease at harvesting. In conclusion, it can be stated that inoculation with Symbivit containing mycorrhizal fungi could be an integral part of spice pepper production.
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Micropropagation of an orchid Dendrobium strongylanthum Rchb.f.
61-64.Views:324A simple and reliable procedure for in vitro propagation of an orchid Dendrobium strongylanthum Rchb.f. was studied. Protochorm was induced from seed explants on 1/2 MS medium supplemented with 0.2 mg/L NAA. A mass of protochorm could be multiplied on proliferated medium of 1/2 Ms containing 0.5 mg/L V-6-BA. And bud differentiation of green global body was cultured in the same media, 2-2.5 cm shoots were formed after 30 day of culture. Addition of mashed banana and 0.5 mg/L NAA to 1/2 MS medium promoted root formation and vigorous growth. The plantlets were acclimatized and transplanted to compound materials of humus:sawdust (1:1) in greenhouse, the survival rate was more than 98%.