Callus Induction and Plant Regeneration from Mature Seeds of Perennial Ryegrass

Objective The aim was to explore callus induction and plant regeneration of perennial ryegrass, as well as provide the foundation for transgenic research on perennial ryegrass.[ Methed] Mature seeds of perennial ryegrass were used as explants to study the effects of different hormone compositions on callus induction, proliferation and plant differentiation. Result The result showed that the induction rate achieved its highest on 2,4-D of 8 mg/L combining with 6-BA of 0.025 mg/L, which was up to 56.42%. Callus were differentiated after two to three generations, the highest differentiation rate 34.14% was achieved in the medium contained MS medium with 6-BA of 2 mg/L, and the differentiation rate was obviously affected by the callus condition after proliferation. The root inducing medium, containing 0.5 mg/L NAA and MS medium with half of macroelement, gained 98% root inducing rate. Conclusien A high frequency genetic regeneration system was established.

In vitro Plant Regeneration of Pepper Cytoplasmic Male Sterility (CMS) Lines via Cotyledon Culture

An in vitro shoot regeneration procedure was developed in pepper （ Capsicum annuum L. ） cytoplasmic male sterility （CMS） lines 9704A and 8214A using cotyledon as explant. The callus and bud cluster derived from cotyledon tissue explants were proliferated on Murashige and Skoog （MS） medium supplemented with different combinations of 6-benzladenine （6-BA）, indole-3-acetic acid （IAA）, gibberellic acid （GA3） and silver nitrate （AgNO3）. From the formula of MS appended with 5.0 mg/L 6-BA, 1.0 mg/L IAA and 5.0 mg/L AgNO3, for the explants callus and bud cluster, the maximum differentiation rates （ respectively 100.0% and 58.3% ） and average number of adventitious bud from each explant （respectively 18.8 and 13.2） were obtained. The optimum medium combination for the elongation of adventitious bud was determined to be： MS ＋ 3.0 mg/L 6-BA ＋ 1.0 mg/L IAA ＋ 5.0 mg/L AgNO3 ＋ 2.0 mg/L GA3, from which the elongation rates of buds from callus and bud cluster were both 100%, and the average number of per explant adventitious bud number reached 6.3 and 5.8, respectively. And all the elongated shoots were successfully rooted on half-strength MS medium supplemented with 0.3-0.5 mg/L IAA.

Establishment of Plant Regeneration System from Immature Embryos of Maize(Zea mays L.) Inbred Lines

[Objective] The aim was to explore the conditions of high frequency induction of embryonic callus and plant regeneration of maize. [Method] Immature embryos of maize inbred lines were used as explants to study the effect of different 2,4-D concentrations on the induction of callus,the effect of different 6-BA concentrations on the differentiation of test-tube plantlet,as well as the effect of different IBA concentrations on the rooting of test-tube plantlet. [Result] 2,4-D showed obvious effect on the induction of inducement rate of maize,and the optimum induction medium was：N6 ＋ 2 mg/L of 2,4-D ＋ 500 mg/L of CH ＋ 500 mg/L of Pro ＋10 mg/L of AgNO3; the optimum differentiation medium was：N6 ＋ 0.5 mg/L of BA ＋ 500 mg/L of Pro; the optimum for the rooting of test-tube plantlet was 1/2 MS ＋ 0.5 mg/L of IBA. [Conclusion] The study had provided basis for the genetic transformation of maize.

Improvement of Plant Regeneration from Immature Embryos of Wheat Infected by Agrobacterium tumefaciens

Wheat, one of the most important food crops, has been extensively studied with respects to plant regeneration and transformation employing the immature embryos as recipient tissues. However, the transformed tissues often become severely necrotic after co-cultivation with Agrobacterium, which is one of the major obstacles in gene delivery. In this study, wheat varieties CB037, Kenong 199, Xinchun 9, Lunxuan 987, and Shi 4185 showed desirable culture potential or high infection ability in Agrobacterium-mediated transformation. Similarly, optimal regeneration conditions were determined by testing their ability to inhibit the cell necrosis and cell death phenotype. Two auxins of 2,4-dichlorophenoxyacetic acid （2,4-D） and 3,6-dichloro-o-anisic acid （dicamba） were evaluated for highly significant effect on both callus and plantlet production, although they were genotype-dependent in wheat. Substitution of 2,4-D by dicamba enhanced the growth and regeneration ability of callus from the immature embryos of most genotypes tested. The callus growth state couldn’t be modified by adding antioxidants such as ascorbic acid, cysteine, and silver nitrate or organic additives such as thiamine HCl and asparagine to the media. On the contrary, the best tissue statement and plant regeneration was achieved by employing the media containing the simplest MS （Murashige and Skoog） components and dicamba without organic components and vitamins. Thereby, our results are thought to inhibit wheat cell necrosis effectively and suggested to be used for more wheat genotypes.

Study on Plant Regeneration of Wheat Mature Embryos Under Endosperm-Supported Culture

To reveal the suitability of using mature embryos as an explant source in wheat tissue culture, mature embryos from eight common wheat cultivars （Triticum aestivum L. cv.） were cultured with or without endosperm to test their efficiency of callus induction and plant regeneration. When embryos were cultured together with endosperm （endosperm-supported culture, ES）, the percentage of callus induction was significantly lower than that when embryos were cultured in the absence of endosperm （non-endosperm-supported culture, NES）. This pattern was evident in most genotypes, regardless of whether 2 or 8 mg L^-1 2,4-D was added in the NES culture. However, in ES culture, more induced calli were differentiated into distinct green spots and they further developed into plantlets. Thus, more plants were regenerated in ES culture than in the NES treatment. Most of the eight tested genotypes showed a significant difference in callus induction rate and plantlet regeneration in both ES and NES cultures. In addition, the enzymatic activity of oxalate oxidase in the callus of ES culture condition was obviously higher than that in the callus of NES culture condition, suggesting that the activity of oxalate oxidase may be a parameter for selection of calli with potential for plantlet regeneration. These results indicate that wheat mature embryos are valuable explants for highly efficient callus induction and plant regeneration, if proper treatment and medium are used.

The Selection of Transgenic Recipients from New Elite Wheat Cultivars and Study on Its Plant Regeneration System

In the protocol of wheat transformation, to use elite wheat cultivars as exogenous gene recipients can speed up the process of commercial field applications of transgenic wheat. However, it is necessary to screen wheat cultivars with good tissue culture response （TCR） continuously from plenty of elite wheat cultivars released for wheat transformation, and it is also important to find a plant regeneration system that is suitable for these cultivars. So, the TCR of mature and immature embryos of six wheat cultivars Chuannong 11 （CN11）, Chuannong12 （CN12）, Chuannong17 （CN17）, Chuannong18 （CN18）, Chuannong19 （CN19）, and Chuannong21 （CN21）, which possess superior agronomic traits, were investigated by using a good TCR wheat cultivar Bobwhite as control. The results indicated that only the immature and mature embryos of CN12, CN17, and CN18 exhibited good TCR compared with Bobwhite. No significant differences were observed between embryos of Bobwhite and of the three cultivars in TCR. Mature embryo-derived calli of CN12 were used as explants for transformation by particle bombardment of SAMDC gene. Seven transformants were obtained and the efficiency was 2.3%. This research supplies three new elite recipient cultivars for wheat transformation. The wheat plant regeneration system used in this research is different from those successful ones reported previously and it could be a reference for other wheat genotypes. Furthermore, Bobwhite and the three wheat cultivars were proved to be 1RS/1BL translocation, by methods of A-PAGE, C- banding, and genomic in situ hybridization （GISH）. These results imply that probably there is some relationship between 1RS/1BL translocation and TCR of wheat embryos. So this research gives us a hint that we should pay more attention to the 1RS/1BL translocations when we screen the wheat cultivars with good TCR and also that the mechanism of the effect of 1RS/ 1BL translocation on TCR is worthy of being investigated.

Dicamba and Sugar Effects on Callus Induction and Plant Regeneration from Mature Embryo Culture of Wheat

To establish a highly efficient plant regeneration system for wheat genetic transformation, the effects of three different concentrations of dicamba and two different sugar types on callus induction and plant regeneration from mature embryo cultures were evaluated. Callus induction and plant regeneration were obtained from mature embryos of two commercial cultivars Zhoumai 18 and Yumai 34 （Triticum aestivum L.） cultured on L3 basal medium. The results showed that the efficiency of mature embryo culture was significantly influenced by the genotypes, sugar types and dicamba concentrations. 4 mg L^-1 dicamba proved the best effective for inducing embryogenic callus and also gave the highest proportion of plants regenerated across the two cultivars. Substitution of maltose by sucrose significantly improved the plant regeneration efficiency in both cultivars. There was a significant interaction between genotype-by-sugar types, and sugar types-bydicamba concentrations. Overall, Zhoumai 18 gave the highest frequency of plant regeneration （82.65%） when dicamba concentration was 4.0 mg L^-1 and with sucrose in initial callus induction. These results will facilitate genetic transformation work with elite wheat.

Plant Regeneration by Callus-Mediated Protocorm-Like Body Induction of Anthurium andraeanum Hort.

This research aims at developing a plant regeneration system from leaf and petiole explants of Anthurium andraeanum Hort., thereby establish a foundation for mass production and transformation. Using tissue culture technique, the conditions for callus induction, protocorm-like body （PLB） formation and plant regeneration from leaf explants and petiole of A. andraeanum, such as basal medium and plant growth regulator, were investigated. Totipotent callus was induced on a 1/2-strength MS medium containing 0.90 μmol L^-1 2,4-dichlorophenoxyacetic acid （2,4-D） and 8.88μmol L^-1 N6-benzyladenine （BA）. The callus exhibited complete hormone autonomy for growth and differentiation of PLBs. This callus proliferated well and was maintained by subculturing on 1/2 MS medium containing 0.90 μmol L^-1 2,4-D and 4.44 μmol L^-1 BA. On average, 8 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred to the 1/2 MS medium with 4.44 μmol L^-1 BA after 8 wk of culture. The regenerated PLBs formed shoots and roots on 1/2 MS medium. After 24 wk of culture on these medium, well-developed plantlets for potting were produced. An efficient micropropagation method was established for indirect PLB formation and plant regeneration from leaf and petiole ofA. andraeanum.

Somatic Embryogenesis and Plant Regeneration from Two Recalcitrant Genotypes of Gossypium hirsutum L.

An improved protocol has been developed for somatic embryogenesis and plant regeneration of recalcitrant cotton cultivars. High callus frequencies and embryogenic tissue were developed in MSB medium supplemented with gradient concentrations of KT and 2,4-D, their concentration decreasing from 0.1 to 0.01 mg·L^-1. Somatic embryos were successfully incubated in 1/2 macronutrient MSB suspension supplemented with 0.5 g· L^-1 glutamine and 0.5 g·L^-1 asparagine. Decrease in macronutrient concentration of MSB significantly alleviated browning and was beneficial to suspension cells. Transformation of somatic embryos into plants was induced in MSB medium supplemented with 3% sucrose, 0.5 g·L^-1 glutamine, 0.5 g·L^-1 asparagine, and 6.0 g·L^-1 agar. The effect of sucrose as carbohydrate was better than that of glucose for plant germination. Using this protocol, regenerated plantlets from the CCRI521 and Zhongzhi86-6 reached to as much as 19.6 and 18.5% somatic embryos, respectively.

Effects of inter-culture, arabinogalactan proteins, and hydrogen peroxide on the plant regeneration of wheat immature embryos

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins （AGPs） and hydrogen peroxide （H202） have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199 （KN 199） and Xinchun 9 （XC9）, together with low-frequency regeneration wheat line Chinese Spring （CS）, presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L-1 AGP or 0.005 to 0.01‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L-1 AGP application level in callus induction medium plant regeneration rates of 8.49,409.06 and 283.16% were achieved for Jimai 22 （JM22）, Jingdong 18 （JD18） and Yangmai 18 （YM18）, respectively; whereas at 100 mg L-1 AGP level, CS （105.44%）, Chuannong 16 （CN16） （80.60%） and Ningchun 4 （NC4） （62.87%） acted the best. Moreover CS （79.05%）, JM22 （7.55%）, CN16 （101.87%）, YM18 （365.56%）, Yangmai 20 （YM20） （10.48%）, and CB301 （187.40%） were more responsive to 0.005 %o of H2O2, and NC4 （35.37%） obtained the highest shoot regeneration rates at 0.01%o of H2O2. Overall, these two methods, inter-culture and AGP （or H2O2） application, can be further applied to wheat transgenic research.

In vitro Plant Regeneration from the Mature Tissue of Navel Orange (Citrus sinensis L. Osbeck) by Direct Organogenesis

An efficient in vitro regeneration system by direct organogenesis from mature nodal and internodal stem segments ofNewhall navel orange (Citrus sinensis L. Osbeck) was developed. Illuminating conditions together with plant growthregulators affected the adventitious bud regeneration frequency and efficiency. The initial 15 d darkness inoculation isbeneficial for the adventitious bud regeneration. The highest regeneration frequency (85.2%) and bud formationefficiency (3.7 per responsive internodal stem segment) were obtained in the media supplemented with 1.0 mg L-1 BAPand 0.5 mg L-1 NAA. ABA at 0.2 mg L-1 positively affected the bud formation efficiency, which amounted to 8.5 buds perinternodal segment in the presence of BAP at 1.0 mg L-1. The adventitious shoots successfully rooted and weretransferred to the soil.

Plant regeneration and genetic transformation in switchgrass——A review

Switchgrass is native to the tallgrass prairie of North America. It is self-incompatible and has varied ploidy levels from diploid（2x） to dodecaploid（12x） with tetraploid and octoploid being the most common. The high yielding potential and the ability to grow well in marginal lands make switchgrass an ideal species as a dedicated biomass producer for lignocellulosic ethanol production. Genetic transformation is an important tool for studying gene function and for germplasm improvement in switchgrass, the genome of which has been sequenced recently. This paper intends to provide a comprehensive review on plant regeneration and genetic transformation in switchgrass. We first reviewed the effect of explants, basal medium and plant growth regulators on plant regeneration in switchgrass, which is a prerequisite for genetic transformation. We then reviewed the progresses on genetic transformation with either the biolistic or Agrobacterium-mediated method in switchgrass, and discussed various techniques employed to improve the transformation efficiency. Finally we reviewed the recent progresses on the use of genetic transformation in improving biomass quality such as the reduction of lignin, and in increasing biomass yield in switchgrass. We also provided a future perspective on the use of new genome editing technologies in switchgrass and its potential impact on regulatory processes.

Advancements in plant regeneration and genetic transformation of grapevine(Vitis spp.)

Grapevine(Vitis spp.)is one of the most economically important fruit crops worldwide,and there is considerable interest in improving its major agronomic and enological traits in response to ever-changing agricultural environments and consumer demands.Molecular genetic techniques in particular,associated with rapid technological advancements,provide an attractive alternative to conventional breeding approaches for developing new grapevine varieties with enhanced yield performance,quality,stress tolerance and disease resistance.To date,several grapevine varieties have been transformed with genes associated with diverse functions through biolistic bombardment and/or Agrobacterium-mediated transformation,and transgenic grape lines have been obtained using established regeneration systems.Nevertheless,a wide range of factors,including genotype,explant source and culture medium,have been shown to affect the efficiency of plant regeneration.Moreover,the selection and use of acceptor materials,bacterial strain and cell density,selectable markers and selection methods also influence transformation efficiency.This paper provides an overview of recent advances in grapevine regeneration and genetic transformation and in-depth discussion of the major limiting factors,and discusses promising future strategies to develop robust plant regeneration and genetic transformation in grapevine.

Enhanced Somatic Embryogenesis and Plant Regeneration from Embryogenic Cultures Derived from Mature Loblolly Pine Zygotic Embryos by Suspension Culture and Medium Selection

Mature zygotic embryos of three families of loblolly pine ( Pinus taeda L.) were cultured on callus induction medium containing 8?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 4?mg·L -1 6 benzyladenine (BA), 4?mg·L -1 kinetin (KT), 500?mg·L -1 casein hydrolysate, and 500?mg·L -1 glutamine for 9 weeks, callus was formed on cotyledons, hypocotyls, and radicles of mature zygotic embryos. Callus was sub cultured on the callus proliferation medium with 1 6?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 0 8?mg·L -1 6 benzyladenine (BA), 0 8?mg·L -1 kinetin (KT) for 9 weeks. White translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESM) and immature somatic embryos was obtained, and the highest frequency of explants forming embryogenic callus was 16 9%. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing embryogenic suspension masses and immature somatic embryos were transferred to medium containing abscisic acid (ABA), polyethylene glycols (PEG), or activated charcoal for enhancing the production of cotyledonary somatic embryos. After mature somatic embryos were cultured on medium containing indole butyric acid (IBA), gibberellic acid (GA 3), BA, and activated charcoal and being lowered sucrose concentration for 4～12 weeks, somatic embryos germinated to form regenerated plantlets. Seventy one regenerated plantlets were transferred to a perlits∶peatmoss∶vermiculate (1∶1∶1) soil mixture, and 23 plantlets survived in the field.

Plant regeneration from protoplasts of hydroxyprolineresistant cell line in Onobrychis viciaefolia

An efficient protocol for plant regeneration from protoplasts of hydroxyproline(HYP)resistant cell line of Onoblychis viciaefolia was established. In SH medium supplemented with 1 mg/L 2, 4-dichlorophenoxy-acetic acid (2,4-D), 0.5 mg/L kinetin (KT) and 0.2 mg/L naphthalene acetic acid (NAA), the division frequency of protoplastderived cells reached uP to over 60 %, and microcalli were obtained in 5-6 wk. Upon transferring them on agar solidified MS medium plus 2 mg/L indole-3-acetic acid (IAA), shoots were induced. After cultivating them on MS medium with or without IAA, roots were regenerated.Chromosome number of all protoplast-regenerated plants examined were normal (2n=28). The protoplast-derived calli and plants grew vigorously on the medium containing 10 mmol/L HYP.

Establishment of highly efficient plant regeneration of Paeonia ostii‘Fengdan’through optimization of callus,adventitious shoot,and rooting induction

Tree peony is a famous ornamental plant,while the low propagation rate is the main hurdles hindering the industry development.Till now,the highly efficient regeneration system for tree peony is not established.In this study,using Paeonia ostii’Fengdan’mature embryos,the effects of variations in inoculation method,initiating culture,adventitious shoot induction,rooting media,plant growth regulators(PGRs),and a nonconventional PGR(plant extracts)on regeneration from explants were evaluated.In embryo cultures,embryonic callus induction rate of 1/4 embryos was the highest among those of embryos with other three technical treatments(whole embryos,1/2 embryos,and pieces of embryos).The woody plant medium(WPM)containing 1.0 mg·L^(-1)6-BA,0.5 mg·L^(-1)GA3,30.0 g·L^(-1)sucrose,and 3.0 g·L^(-1)phytagel significantly improved shoot induction and multiplication.3.0 mg·L^(-1)plant extracts promoted hypocotyl germination,rooting,and root growth,in direct embryo culture,and a combination of 3.0 mg·L^(-1)plant extracts+2.0 mg·L^(-1)IBA+1.5 mg·L^(-1)IAA produced optimal rooting induction rate for multiple shoots in direct embryo culture and indirect somatic embryogenesis.For the three in vitro micropropagation methods,the highest shoot proliferation coefficient(5.4±0.2)was obtained with indirect somatic embryogenesis.Fortunately,the propagation ability of shoots remains high,even when culture propagation was continued for more than two years.Thus,a reliable system for plant regeneration from mature embryos derived from P.ostii’Fengdan’callus and two direct embryo culture systems have been established.The novel regeneration system could facilitate uniform seedling production.

Plant regeneration from cultured protoplasts of a glutinous rice

Young embryos of rice (Oryza saliva L. subsp. japonica var. Guo-xiang No.l) were cultured on MS agar medium(2,4-D 2 mg/l). Calli were formed and subcultured on N6 agar medium (2,4-D 2 mg/l). After selection, the small, grainy and pale yellowish cell clusters with dense cytoplasm were used in protoplast preparation. Isolated protoplasts were cultured in N6 medium (2,4-D 1 mg/l, 6-BA 0.2 mg/l)1 with agarose block culture method. The protoplasts grew, divided and formed calli. After inducing differentiation, the regenerated mature plants were obtained.

Plant Regeneration from In Vitro Cultured Hypocotyl Explants of Euonymus japonicus Cu zhi

Adventitious shoots were successfully regenerated from hypocotyl explants of in vitro cultures of Euonymus japonicus Cu zhi. Hypocotyl slices were cultured on Murashige and Skoog （MS） and B5 basal medium supplemented with varied concentration of different plant growth-regulators, e.g., α-naphthaleneacetic acid （NAA） and indole-3-butyric acid （IBA） in combination with 6-benzylaminopurine （6-BA） and kinetin. The study showed that shoots could be directly regenerated from hypocotyl explants without the intervening callus phase; MS medium was more suitable for adventitious shoots regeneration. The ability of hypocotyls segments to produce shoots varied depending upon their position on the seedlings. The highest regeneration rate was obtained with hypocotyl segments near to the cotyledon cultured on MS basal medium supplemented with 1.5 mg L^-1 6-BA and 0.05 mg L^-1 NAA （63.64%）. The regenerated shoots were readily elongated on the same medium as used for multiplication and rooted on half-strength MS basal medium supplemented with 1.0 mg L^-1 IBA and 100 mg L^-1 activated carbon. After being transferred to greenhouse conditions, 96% of the plantlets were successfully acclimatized. This regeneration system is applied for genetic transformation now.

Direct Adventitious Bud Induction and Plant Regeneration of Rosa hybrida Samantha

Effect of explant, site of leaflet, induction period in the dark and combinations of plant growth regulators on direct adventitious bud induction and plant regeneration of Rosa hybrida Samantha was investigated. The results showed that after an induction period of 8 d on MS medium with 1.5 mg L-1 TDZ and 0.05 mg L-1 NAA in the dark and a subculture on MS medium with 0.5 mg L-1 BA and 0.01 mg L-1 NAA under light, the best plant regeneration was obtained and the regeneration frequencies of leaflets and petioles were 51.8 and 10% respectively. There was no significant difference in regeneration ability between leaflets at different sites of the compound leaves, longer time of induction in the dark or high concentration of auxin would cause callus formation, which was disadvantageous for shoot regeneration, and the regeneration frequency was significantly reduced. This regeneration system could be applied for genetic transformation of this cultivar in the future.

Tissue Culture and Plant Regeneration of Chicory

[ Objecllve] To establish a high-frequency regeneration system of chicory （ Cichodum intybus L. ） using leaf segments of aseptic seed- lings. E Method] Calluses and adventitious buds of chicory were induced by inoculating explants on MS medium supplemented with 6-BA （6-benayl aminopurine） and NAP, （naphthylacetic acid） at different final concentrations. [ Result] When lower part of leaves derived from 20-day-old seedlings was used as explant and inoculated on MS medium containing 2.0 rng/L 6-BA, 0.5 mg/L NAA and 40 g/L sucrose, the frequency of adventitious bud formation was 90.0%. When the regenerated shoots were cultured in 1/2 MS medium containing 0.1 mg/L NAA, the frequency of root forma- tion was 88.3%. All rooted plants transplanted in pots could survive and grew well without abnormal shape. [ Conclusion] Better differentiation of adventitious buds can be achieved by inoculating the lower part of leaves derived from 20-day-old seedlings on MS medium containing 2.0 mg/L 6- BA, O. 5 mg/L NAP, and 40 g/L sucrose. The 1/2 MS medium containing O. 1 mg/L NAP, is most suitable for rooting.