Growth and Transcriptomics Analysis ofMichelia macclurei Dandy Plantlets with Different LED Quality Treatments

Michelia macclurei Dandy is a significant tree species that has extensive cultivation for forestry and horticulture purposes in southern China,owing to its economic and practical importance.Light quality influences plantlet growth and development during tissue culture.However,the growth characteristic and molecular regulation of M.macclurei under different light quality conditions are not well understood yet.In this study,we investigated the morphological,chlorophyll content,and transcriptomic responses of M.macclurei plantlets under different light-emitting diode(LED)qualities,including white,blue,and red light.The results showed that blue light significantly increased plant height(21.29%)and leaf number(18.65%),while red light decreased plant height and leaf number by 7.53%and 16.49%,respectively.In addition,the plantlets’chlorophyll content and etiolation rate were significantly reduced by blue and red light quality compared to white light.Compared to white light,blue light had a negative effect,leading to decreased rooting rate(64.28%),root number(72.72%),and root length(75.86%).Conversely,red light had a positive effect,resulting in increased rooting rate(24.99%),root number(109.58%),and root length(72.72%).Transcriptome analysis identified 54 differentially expressed genes(DEGs)in three groups that consisted of blue light vs.white light(BL-vs-WL),red light vs.white light(RL-vs-WL),and red light vs.blue light(RL-vs-BL).Specifically,21,7,and 41 DEGs were identified in the three groups,respectively.The DEGs found in the RL-vs-WL and BL-vs-WL groups were involved in plant hormone signaling,nitrogen metabolism,and phenylpropanoid biosynthesis pathways,which suggests that M.macclurei plantlets adapt to the changes of light quality via modulating gene expression.Overall,our study provides valuable insights for understanding the molecular and morphological responses of M.macclurei plantlets under different light qualities.

Evaluation of Different Substrates Compositions for Acclimatization of Tissue Culture Taro Plantlets in a Propagator

Taro is cultivated in most Regions of Cameroon and it is affected by taro leaf blight disease since 2010 which has decreased its production. Lack of disease-free planting materials has been a main problem to farmers. This study was carried out at International Institute of Tropical Agriculture (IITA) Yaounde and Institute of Agricultural Research for Development (IRAD) Bambui to assess different substrates for acclimatization of tissue culture taro plantlets in apropagator. No information is available on acclimatization of Cameroonian taro plantlets in different substrates. Taro plantlets from tissue culture were acclimatised in a propagator for six weeks under different substrates, the first substrate consisted of sterile three parts of soil and one part of river sand mixed together (3:1), the second substrate consisted of sterile two parts of soil and two parts of river sand mixed together (2:2), the third substrate consisted of sterile two parts of soil, one part of rice husk and one part of river sand mixed together (2:1:1) and the fourth substrate consisted of sterile one part of soil and three parts of river sand mixed together (1:3). After acclimatisation of the different taroplantlets (Dark green petiole with small leaves (L1), Red petiole with small leaves (L2), Light green petiole with large leaves (L3) and Light green petiole with small leaves (L4) in these four substrates, it was observed that the best growth rate of plant was recorded on substrate sand + soil (1:3). The other substrates showed moderate growth of plants. Substrate sand + soil (1:3) can be recommended for acclimatization of Cameroonian taro plantlets.

Effects of different LEDs light spectrum on the growth,leaf anatomy,and chloroplast ultrastructure of potato plantlets in vitro and minituber production after transplanting in the greenhouse

Light spectrum plays an important role in regulating the growth and development of in vitro cultured potato(Solanum tuberosum L.) plantlets. The status of potato plantlets at the end of in vitro stage influences the minituber production after transplanting. With 100 μmol m^-2s^-1 total photosynthetic photon flux density(PPFD), a light spectrum study of 100% red light emitting diodes(LEDs) light spectrum(RR), 100% blue LEDs light spectrum(BB), 65% red+35% blue LEDs light spectrum(RB), and 45% red+35% blue+20% green LEDs light spectrum(RBG) providing illumination at the in vitro cultured stage of potato plantlets for 4 weeks using fluorescent lamp as control(CK) was performed to investigate the effects of LEDs light spectrum on the growth, leaf anatomy, and chloroplast ultrastructure of potato plantlets in vitro as well as the minituber yield after 2 months transplanting in the greenhouse. Compared to CK, RB and RBG promoted the growth of potato plantlets in vitro with increased stem diameter, plantlet fresh weight, plantlet dry weight, and health index. Furthermore, BB induced the greatest stem diameter as well as the highest health index in potato plantlets in vitro. Root activity, soluble protein, and free amino acid were also significantly enhanced by BB, whereas carbohydrates were improved by RR. In addition, thickness of leaf, palisade parenchyma and spongy parenchyma was significantly increased by BB and RBG. Chloroplasts under BB and RBG showed well-developed grana thylakoid and stroma thylakoid. Unexpectedly, distinct upper epidermis with greatest thickness was induced and palisade parenchyma and spongy parenchyma were arranged neatly in RR. After transplanting in the greenhouse for 2 months, potato plantlets in vitro from BB, RB, and RBG produced high percentage of large size tuber. BB improved fresh and dry weights of the biggest tuber but decreased tuber number per plantlet. In addition, RBG increased tuber number as well as tuber fresh and dry weight slightly. Our results suggested monochromatic blue LEDs as well as combined red, blue or/and green LEDs light spectrum were superior to fluorescent lamp spectrum in micro-propagation of potato plantlets. Therefore, the application of RBG was suitable;BB and RB could be used as alternatives.

Transcriptome analysis reveals effects of red and blue lightemitting diodes(LEDs)on the growth,chlorophyll fluorescence and endogenous plant hormones of potato(Solanum tuberosum L.)plantlets cultured in vitro

Red and blue light illumination has been reported to significantly affect plantlet growth.Potato is an important food and feed crop in the world and potato plantlet cultured in vitro plays an important role in potato production.However,few studies have documented the effects of red and blue light on the growth of potato plantlets revealed at the transcriptome level.The objective of this study was to determine the growth and physiological responses of potato plantlets cultured in vitro under monochromatic red(RR),monochromatic blue(BB)as well as combined red and blue(RB)LEDs using the RNA-Seq technique.In total,3150 and 814 differentially expressed genes(DEGs)were detected in potato plantlets under RR and BB,respectively,compared to RB(used as control).Compared to the control,the DEGs enriched in"photosynthesis"and"photosynthesis-antenna proteins"metabolic pathways were up-regulated and down-regulated by BB and RR,respectively,which might be responsible for the increases and decreases of maximum quantum yield(F_(v)/F_(m)),photochemical quantum yield(φ_(PSII)),photochemical quenching(q_(P))and electron transfer rate(ETR)in BB and RR,respectively.Potato plantlets exhibited dwarfed stems and extended leaves under BB,whereas elongated stems and small leaves were induced under RR.These dramatically altered plantlet phenotypes were associated with variable levels of endogenous plant hormones gibberellin(GAs),indoleacetic acid(IAA)and cytokinins(CKs),as assessed in stems and leaves of potato plantlets.In addition,monochromatic red and blue LEDs trigged the opposite expression profiles of DEGs identified in the"plant hormone signal transduction"metabolic pathway,which were closely related to the endogenous plant hormone levels in potato plantlets.Our results provide insights into the responses of potato plantlets cultured in vitro to red and blue LEDs at the transcriptomic level and may contribute to improvements in the micro-propagation of potato plantlets cultured in vitro from the light spectrum aspect.

Effects of Sion Cold Resistance of Cymbidium Plantletsin vitro

To determine the medium with the concentration of Na2SiO3 , the effect of different concentrations of Na2SiO3 on cold resistance of Cymbidium plantlets in vitro was studied. The results showed that Si enhanced osmotic regulation ability of Cymbidium evidently on cold resistance with increasing contents of free Pro, soluble sugar, soluble protein and decreasing contents of MDA. Compared with CK, osmotic regulation substances are accumulated rapidly. Free Pro, soluble sugar, soluble protein, MDA could be adopted as reference indices to evaluate capability of frost resistance of Cymbidium. By experiment we stated Na2SiO3 (3mmol/L) could relieve low temperature injury of Cymbidium plantlets in vitro. Mitigative effect was reduced with increasing concentration of Na2SiO3 .

Generation of Virus Free Potato Plantlets through Meristem Culture and Their Field Evaluation

Different aspects of micropropagation through meristem culture for the production of virus indexed source plants, in vitro tuberization and field evaluation of the in vitro regenerated plants were studied on four commercial cultivars of potato (Solanum tuberosum L.) viz., Diamant, Cardinal, Shilbilati and Lalpakri. The investigation was conducted at Rajshahi, Bangladesh from December 2010 to March 2012 to produce virus-free potato plantlets through meristem culture, shoot multiplications with root induction as well as their acclimatization and evaluation of morphological characters and tuber yield under field condition. Shoot tips of 25 - 30 day old field-grown plants of above mentioned four cultivars were used for meristem isolation. After isolation, meristems of these varieties of potato were cultured on “M” shaped filter paper bridge in Murashige and Skoog (MS) liquid medium. Four different treatments of media formulations viz. 0.1 mg/L KIN + 0.1 mg/L GA3, 0.1 mg/L KIN + 0.5 mg/L GA3, 0.5 mg/L KIN + 0.1 mg/L GA3 and 0.5 mg/L KIN + 0.5 mg/L GA3 were used as plant growth regulators. From these formulations MS + 0.1 mg/L KIN + 0.5 mg/L GA3 was found to be the best for the primary establishment of meristem culture. The primarily established meristems were subcultured on to MS semisolid basal medium supplemented with four different treatment combinations of hormones viz. 0.5 mg/L BA + 1.0 mg/L IBA;0.1 mg/L KIN + 0.1 mg/L GA3;0.5 mg/L BA + 0.5 mg/L GA3 and 0.5 mg/L KIN + 0.5 mg/L GA3 were used to identify the suitable media compositions for shoot proliferation. Results showed that out of these four media treatments the formulation 0.5 mg/L BA + 0.5 mg/L GA3 was found to be the best suitable for shoot generation. Among the four cultivars of potato higher frequency of shoot proliferation (number of shoots/explant and longest shoot length) was observed in Diamant, though the highest shoot formation (76%) was recorded in Cardinal. Virus free in vitro grown potato plantlets were obtained through DAS-ELISA test and used substantially for micro-propagation. After gradual acclimatization of rooted plantlets of four potato cultivars, they were transferred into the field for cultivation and established successfully. It was observed from the field study of in vitro meristem-derived plantlets that there were no virus-affected plants. The virus-free exotic varieties were much superior in all vegetative attributes and yield compared to those of indigenous varieties with producing potato plants of normal height. In contrast, the indigenous varieties took a longer time to tuber initiation and maturity, lower plant height and number of leaves per plant, a higher number of tubers but a lower amount of tuber weight per plant, and poorer tuber grade than the exotic varieties. However, the variety Cardinal exposed the best performances in the context of survival percentage of plantlets (90%), days to tuber initiation (DTI), a number of leaves per plant (NL), tuber weight per plant (343.40%) and the percentage of rich tuber grade.

Study on Efficient Multiplication of Blueberry(Vaccinium corymbosum)Plantlets in Swinging type Temporary Immersion Bioreactors System(S-TIBs)

The swinging type temporary immersion bioreactors system （S-TTBs） is a kind of new and advanced method of tissue culture. The efficient multiplica- tion technology of blueberry （ Vaccinium corymbosum） plantlets in S-TTBs was systematically studied through L9 （3^4） orthogonal experiment with three factors （culture medimn volmne, swing angle, and inoculation density） and completely randomized experiment with two factors （swing frequency and immersion time）. The results showed that the optimal culture parameters were set as follows： the culture medium volume of 250 nil/bottle, the swing angle at 45 ° , and the inoculation density of 60 plantlets/bottle. The optimal swing frequency was 1 time/6 h, and the immersion time was set as 60 s.

Effects of Non-Conventional Substrate and NPK Fertilizer on Cassava Plantlets Acclimatization for Plant Material Production

The adaptation of cassava plantlets to natural conditions remains the main obstacle in the process of free virus plantlets production. In this study, cassava plantlets acclimatization was assessed by pre-acclimatization, height of acclimated plantlets, substrate, and nutrient watering solution. The results revealed that pre-acclimatization gave a high survival rate (61.9%) whereas the direct tunnel acclimatization was conducted to low rate (27.31%) of plantlets. Furthermore, the height of plantlets influenced significantly (p < 0.05) their survival rate with best survival rate observed on plantlet height from 1 cm to 5 cm. There was no significant difference between the substrates used and watering solutions. However, the Sawdust substrate gave a high survival rate (46.67%) and better phylogenesis (1.8) in the variety RB 89509. Likewise, plantlets treated with NPK nutrient watering solutions (20-10-10) had a better survival rate (48.57%), better phylogenesis (2.125) with the variety RB 89509 and better growth in height (1.18 cm) with the variety BF92/0267. These findings constitute a database for the establishment of the technical itinerary for the acclimatization of cassava plantlets.

Influence of Watering Solution and Phenotype on the Growth of <i>in Vitro</i>Propagated Pineapple (Smooth Cayenne Cultivar) Plantlets during Acclimatization

Pineapple is the first fruit crop cultivated in south Benin that greatly contributes for food and nutritional security and farmers’ income. But the lack of homogenous planting material constitutes the major constraint for improving pineapple yield. In vitro micropropagation is now used in the production of homogenous and free disease planting materials of pineapple. However, the acclimatization to natural condition of pineapple plantlets is an important step in planting material production of this crop. Here, we determined the intrinsic and extrinsic factors which influence the behavior of plantlets during the acclimatization process. For this purpose, plantlets from different categories were selected, trimmed and planted on a horticultural substrate made up of potting soil, white sawdust and compost previously sterilized. The plantlets were then incubated in under acclimatization greenhouse with average temperature of 29˚C and 70.2% of humidity. A batch of plantlets was subjected to two different watering solutions: Shive and Robbins solution and NPK 14-6-5 foliar fertilizer. The results obtained initially showed high rate (100%) of survival and growth of the plantlets watered with Shive and Robbins solution against 50% of the plantlets watered with the foliar fertilizer solution. In addition, the plantlets with spread pores exhibiting the characteristics of which the number of leaves varies between 9 and 11, the weight between 1.2 and 1.5 g, the size of 4.5 to 5.5 cm, and a good junction between the aerial part and the root system were those which were successfully grown in acclimatization phase under greenhouse, unlike plantlets with erected pores having lower success rate. This study goes a long way in providing good procedures of acclimatization of homogenous and free disease planting material of pineapple to the famers.

Effect of desiccation on regeneration frequency of green plantlets and some biochemical properties of calli of wild rice

Wild rice is an important resource of usefulgenes to rice breeders. However, low regener-ation frequency is an obstacle to use the valu-able genes. We used desiccation to improve theregeneration frequency and studied the bio-chemical changes of calli of wild rice after des-iccation.Materials used in this experiment werewild species O. rufipogon, O. meyeriana, O.alta, and O. brachyantha. Young panicles(0.1-0. 5 cm in length of the inflorescence)

Effect of mini-cutting size on adventitious rooting and morphophysiological quality of Ilex paraguariensis plantlets

As the energizing and therapeutic effects of infusions of mate(Ilex paraguariensis A.St.-Hil)leaves are increasingly recognized,rising global consumption is also increasing the demand for plantlets with high genetic and morphological qualities.Mini-cuttings are an alternative for clonal plantlets;however,more details are needed on factors that affect the success of this technique.Here,toward maximizing production,we evaluated adventitious rooting competence of mini-cuttings of different sizes and the morphophysiological quality of the resultant plantlets.Small(one bud;length up to 2.5 cm),medium(multiple buds;length 2.6–5.0 cm),and large(multiple buds;length 5.1–10.0 cm)mini-cuttings were planted in a mixture of commercial substrate,vermiculite,and coarse sand(1:1:1 v:v:v),grown in a humidity chamber,then evaluated after 45,60,75,and 90 days for survival,percentage of callus and shoot formation,number and length of shoots,percentage of rooted cuttings,and number and length of roots.Any rooted mini-cuttings were then cultivated in commercial substrate and subsurface soil(2:1 v:v)and evaluated after 30,60,90 and 120 days of cultivation for morphophysiology of the plantlets,percentage of survival,number of leaves,shoot height,stem diameter,and ratio of shoot height to stem diameter.After 120 days,the plantlets were evaluated for the ratio of shoot and root dry mass,total length,surface area,total volume,and mean diameter of roots and Dickson quality index.Adventitious rooting of the mini-cuttings and the morphophysiological quality of the plantlets were not influenced by the size of the vegetative propagule.Therefore,single-bud mini-cuttings have similar adventitious rooting competence,and result in plantlets with adequate morphophysiological quality,based upon leaf development,shoot height,stem diameter and length,and surface area,volume and diameter of roots.Single-bud mini-cuttings up to 2.5 cm long can thus be used to maximize production of mate plantlets by mini-cuttings.

Construction and Characterization of cDNA Library from Water-Stressed Plantlets Regenerated in vitro of Populus hopeiensis

In order to isolate and clone water-stress-responsive genes, total RNA was extracted from water-stressed plantlets regenerated in vitro of Populus hopeiensis using a QIAGEN RNeasy Plant Mini Kit. CDNA, synthesized by LD-PCR with the SMART cDNA Library Construction Kit, was in vitro packaged into a phage λTriplEx2 vector. The resulting primary library and amplified library have a titer of 1.68×10^6 and 1.69×10^9 pfu·mL^-1 respectively. The combination ratio reached 98.8% and the average size of inserts was about 800 bp. In addition, the percentage of inserted fragments （〉400bp） was approximately 90%. The results indicate that a cDNA library has been successfully constructed.

Recovery of somatic embryos and plantlets from callus of mature masson pine embryos

Remarkable progress has been made in the development of systems for somaticembryogenesis in conifers since 1985. Currently however, it is still recalcitrant to inducesomatic embryo development from Pinus species. It is now only possible to obtain somaticembryos and plantlets from Pinus caribaea. P. lambertiana and P. tadea through in vitroembryogenesis. Of the three Pinus species which have expressed embryogenesis in explant-derivedcultures. only P. lambertiana has been successfully induced to produce somatic embryos frommature zygotic embryos.

Obtaining of Haploid Plantlets of Chinese Scholartree (Sophora japonica L.) by Anther Culture

During the past years much progress has been made in the anther culture of woody plants. The potential of using haploid plants and homozygous lines in plant breeding programs has long been recognized, especially in woody plants. However, the conventional method employed by plant breeders for their production is cumbersome, laborious and not very efficient. Using in vitro culture method, haploids can be obtained in a matter of weeks or months, and by doubling their chromosome number homozygous

枸杞花药培养的初步研究(英文)

To investigate the culture technique in anther of Chinese wolfberry,we optimized the culture medium(including hormone combination)and culture conditions.The results showed that calluses were induced from all the six tested Chinese wolfberry materials,but the induction rate of callus varied toward the materials with different genotypes.When the experimental materials were cultured on medium appended with 2,4-D 1.0 mg/L and KT 1.0 mg/L under dark,the callus induction rate reached 20.0 % in this study,and this hormone combination should be the optimum for anther culture of Chinese wolfberry.With MS appended with 6-BA 0.5 mg/L and NAA 0.1 mg/L as differentiation medium and that appended with NAA 0.1 mg/L,the plants could be yielded in 20 days.

诸葛菜试管花朵发育的研究(英文)

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NaCl胁迫对甘薯试管苗生长和离子含量的影响(英文)

[Objective] This study aimed to investigate the salt-tolerance mechanism of sweet potato.[Method]Two sweet potato varieties of Xu 25-2(salt-tolerant cultivar)and Triumph 100(salt-sensitive cultivar)were treated by sodium chloride with the concentration of 0 mmol/L and 100 mmol/L.After 20 days,Na+ content and Na+/K+ ratio in the roots,shoots and leave were determined by the flame photometer,while dry weight and fresh weight of roots,shoots and leave in different varieties were also studied.[Result]The growth of two sweet potato varieties was inhibited under salt stress,so the plant became shorter,leaf and root became fewer,dry weight of roots and leave decreased,but seedlings of Xu 25-2 were inhibited slightly.Furthermore,Na+ content and Na+/K+ ratio in roots,shoots and leaves of two sweet potato varieties increased.Na+ content of salt-tolerant Xu 25-2 was low in roots,shoots and leaves,while Na+ content of salt-sensitive Triumph 100 was high in shoots and leave of seedlings,but the change range of Xu 25-2 was less than that of Triumph 100.[Conclusion]The lower Na+ content and Na+/K+ ratio in leaves under salt stress were the most important characteristics for salt-tolerance of sweet potato varieties.

In vitro callus induction and plantlet regeneration of Achyranthes aspera L.,a high value medicinal plant

Objective:To study callus induction from different explants(internode,leaf,root)and in vitro plantlets propagation from medicinally important plant Achyranthes aspera L.Methods:Sterilized explants were prepared by uning 0.1%HgCl_2 and 0.5%Bavistin and callus was obtained when cultured onto Murashige Skoog's(MS)medium by using different concentrations and combination of 2,4-D.NAA.BAP,IAA,IBA with 3%sucrose and 0.8%agar.Induced callus was immediately transferred to MS medium containing at different concentrations of phytohormones for shootlets and rootlets induction respectively.Results:Sterilization treatment of 0.1%HgCl_2.for 2-3 min and Bavistin 0.5%for 10-12 min showed the highest percentage of asepsis and survival rate.Maximum induction of callus was obtained from a combination of 2.0 mg/L 2,4-D and 0.5 mg/L NAA from leaf.Highest shootlets number(4.83±0.l7)and length(3.8±0.16)cm were observed on full strength MS medium when fortified with BAP 4.0 mg/L and KIN 0.5 mg/L.Concerted efforts of BAP 10 mg/L and NAA 0.5 mg/L on full strength MS medium showed highest leaf number(6.77±0.94).In vitro raised shoots were allowed to root on different strengths of MS medium fortified with IAA and IBA at different concentrations.Experimentally,3.0 mg/L IBA was enabled to induce maximum rootlets number(10.0±9.82)on full strength MS medium.Afterwards,regenerated shoots with well developed roots were successfully subjected to hardening process and were acclimatized.The survived plantlets showed 66.67%survival frequency without any morphological abnormality.Conclusions:The results demonstrated that different explants were good source of callus induction,morphology analysis as well as indirect plantlets regeneration.

Influencing in vitro clonal propagation of Chonemorpha fragrans(moon) Alston by culture media strength,plant growth regulators,carbon source and photo periodic incubation

Chonemorpha fragrans is an endangered medicinal woody climber,regarded among alternative plant sources of camptothecin.Camptothecin is a monoterpene indole anti-cancer alkaloid with annual trade value of over three billion U.S.dollars in the recent,and is used in the production of its analog drugs approved for the chemotherapy of cancer of varied types.Effects of plant growth regulators,culture media strength and photoperi-odic duration on the micropropagation ef ciency of C.fragrans from nodal segment explants were studied on Murashige and Skoog(MS)medium amended with Thidiazuron(TDZ),Benzylaminopurine(BAP)or Kinetin(Kin).Thidiazuron was more ef cient over BAP and Kin when half basal MS medium was used over full or quarter strength.Results of carbon source experiment showed sucrose as the most effective over glucose,fructose,and maltose in the clonal production.Studies on the photope-riodic incubation duration showed 12 h as the best light period and sub or supra-optimal resulted in the production of abnormal and albino micro shoots.Experimental results on the evaluation of physiological,biochemical parameters showed the role of pigment molecules and antioxidant systems in the production of albino micro shoots.

A rapid multiplication system for'Candidatus Liberibacter asiaticus'through regeneration of axillary buds in vitro

‘Candidatus Liberibacter asiaticus(CLas)’,which causes citrus Huanglongbing(HLB)disease,has not been successfully cultured in vitro to date.Here,a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected‘Changyecheng’sweet orange(Citrus sinensis Osbeck).Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds.A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination(DAG).This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse.To minimize contamination during in vitro regeneration,CLas-infected axillary buds were micrografted onto seedlings of‘Changyecheng’sweet orange and cultured in a liquid medium.In this culture,the titers of CLas in regenerated shoots rapidly increased from 7.5×10^(4)to 1.4×10^(8)cellsμg^(-1)of citrus DNA during the first 40 DAG.The percentages of shoots with>1×10^(8)CLas cellsμg^(-1)DNA were 30 and 40%at 30 and 40 DAG,respectively.Direct tissue blot immunoassay(DTBIA)indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse.The disease symptoms in the plantlets were die-back,stunted growth,leaf necrosis/yellowing,and defoliation.The death rate of the plantlets was 82.0%at 60 DAG.Our results show that CLas can effectively multiply in citrus plantlests in vitro.This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.