Induction of Somatic Embryogenesis and Long Term...

625Current Trends in Biotechnology and PharmacyVol. 7 (2) 625-634 April 2013, ISSN 0973-8916 (Print), 2230-7303 (Online)

AbstractWe have been successful in the long term

maintenance of embryogenic cultures for twocultivars of litchi (Litchi chinensis Sonn.) Purbiand Bedana. The embryogenic lines could bestably maintained through frequent sub-culturingon long term maintenance medium containing2,4-D + IASP without loss of embryogeniccompetence. The combination of activatedcharcoal, sucrose, and polyethylene glycol in theembryo development and maturation mediumprovided the highest number of mature embryosthat were not very rich in starch granules but hadhigh phenolic compounds. Nevertheless thematured somatic embryos were successfullygerminated on MS medium consisted of MS saltsand B5 vitamins with Kinetin 1 mgl-1, Gibberellicacid (GA) 5 mgl-1, coconut water 50 ml l-1, 3%(w/v) sucrose, 7 gl-1 agar and grown into normalplants on conversion medium (CM) and itsefficiency was different in different cultivars i.e.in Purbi-49% and Bedana-36.1%.

Keywords: Litchi chinensis, litchi, embryoconversion, embryo germination, embryoregeneration, embryogenesis

IntroductionThe development of an efficient regenera-

tion system is a prerequisite for transformationand production of genetically modified cropplants. Successful embryogenesis and theirconversion to litchi plants have been restrictedto a few species including Indian cultivars Litchichinensis cv Purbi, Litchi chinensis cv Bedana

and Litchi chinensis cv Shahi etc. or hybridsinvolving one of these species (1, 2, 3, 4). Severalauthors have attempted to induce somaticembryogenesis from vegetative tissues of litchi,such as leaves (5, 6), zygotic embryos (1), andanthers (3). Cell and protoplast culture wasreported in Longan (7). In vitro techniques, forthe culture of protoplasts, anthers, microspores,ovules and embryos have been used to createnew genetic variation in the breeding lines, oftenvia haploid production. Cell culture has alsoproduced somaclonal and gametoclonal variantswith crop-improvement potential. The culture ofsingle cells and meristems can be effectivelyused to eradicate pathogens from plantingmaterial and thereby dramatically improve theyield of established cultivars (8). Targetingspecific trait gene is predicated upon the abilityto regenerate elite selections of what aregenerally trees from cell and tissue cultures. Theintegrity of the clone would thereby remainunchanged except for the altered trait. Thisregeneration system can be utilized for thegenetic transformation of perennial tropical andsubtropical fruit crops i.e. Longan (Dimocarpuslongan Lour) and Litchi (Litchi chinensis Sonn.)(9). In most of the important fruit crops, tissueculture is well established for plant regenerationeither via somatic embryogenesis ororganogenesis. Since somatic embryo culturesoften originate from a single cell, it is an idealsystem for induction of mutations as it helps inpreventing chimerism. The rate of somaticembryo germination is very poor, which hasbecome a major hurdle for large-scale plant

Induction of Somatic Embryogenesis and Long TermMaintenance of Embryogenic Lines of Litchi

D.K. Das* and A.RahmanPost Graduate Department of Biotechnology, T.M. Bhagalpur University, Bhagalpur-812007

*For Correspondence - [email protected]

Induction of somatic embryogenesis and long term


multiplication of desirable induced mutants. Themajor focus will be on the development of mutantlines of fruits with improved tolerance againstabiotic and biotic stresses, seedlessness e.g.Litchi, Longan, Guava and Citrus (10).Biotechnology for longan and litchi has beenresearched for the last 20 years. At first, cellengineering based on tissue culture wasemphasized, including: 1. Saving cultures withyoung and small embryos 2. Anther or pollenculture to obtain haploid plants and 3. Basicresearch focused on protoplast culture.Subsequently, with the rapid development ofbiotechnology, research was re-oriented towardgenetic engineering, including mainly molecularmarker techniques and new germplasmconstruction by protoplast fusion andtransformation (11). The long term maintenanceof embryogenic cell lines has not been reportedin litchi. Since the reported embryogenicregeneration protocol of litchi which is highlyefficient obtained from zygotic embryos is of longduration (9-12 months) and availability of explants(zygotic embryos) once a year so thatdevelopment of long-term maintenance of celllines is very important for mass propagation andcrop improvement of litchi. However, the longterm maintenance of cell lines is highly genotypedependence in litchi like grape which has beenreported using mainly liquid medium (12, 13).Embryos produced via liquid culture, often yieldmorphological abnormal plantlets in litchi likegrape (14). These limitations hinder the possibleutilization of long-term solid or liquid embryogeniccell suspension cultures for genetictransformation in litchi like grapevines. Thedifficulties in induction of somatic embryogenesis,the low reproducibility of the results and thenarrow temporal availability of explants meanthat, once induced and established, theembryogenic cultures are very valuable materialin litchi like grape (15). Under certain conditions,litchi like grape, embryogenic cultures have beenmaintained almost indefinitely constituting a long-term source of somatic embryos (16, 17). In orderto study factors affecting the long-termmaintenance of embryogenic lines cultured on

solid media, we are reporting a method to induceand maintain the embryogenic cultures in twoIndian genotypes i.e. Purbi and Bedana of litchi.We further report for the first time in litchi thatthese lines can be maintained for long time andthat they can be readily converted into normalplants during that period.

Materials and MethodsCulture initiation: To establish embryogeniccultures, young immature Indian litchi fruits ofboth cultivars i.e. Purbi and Bedana werecollected from Horticulture Division, BiharAgricultural University, Sabour, Bhagalpur (India)stored in plastic bags at 4OC for approximately24 h. Prior to dissection, fruits were disinfectedwith a 1.3% sodium hypochlorite (NaOCl) solutioncontaining 0.1 % (v/v) Tween 20 for 10 min andrinsed 4 times with sterile distilled water (5 min/rinse). The sterilized immature fruits wereexcised and isolated zygotic embryos weretransferred to medium for callus induction andregeneration following the protocol of (1). In theculture initiation only two litchi cultivars i.e. Purbiand Bedana were taken for development ofembryogenic lines. This medium containingNN(18) salts supplemented with 30 gl-1 sucrose,100 mgl-1 myoinocitol, 500 mgl-1 caseinhydrolysate, 17 µM indole-3-acetyl-L-asparticacid (IASP, dissolved in dimethyl sulphoxide), 9µM 2,4-dichlorophenoxyacetic acid (2,4-D) and1 µM 6- benzylaminopurine (BAP). The mediumpH was adjusted to 5.6 prior to autoclaving and7.0 gl-1 agar were added as the gelling agent.The medium was autoclaved for 20 min at 1210C.Both 2, 4-D and BAP were added beforeautoclaving and IASP was added afterautoclaving. The medium was dispensed into 100× 15 mm disposable petri plates (30 ml per plate).Fifteen zygotic embryos were inoculated to eachplate in a total of four replicated plates per cultivar.These plates were then incubated in the dark at25 ± 20C until callus developed (ca. 45 days). Allculture vessels (Petri dishes, Jars) were sealedin aseptic condition.

Long term maintenance of embryogeniclines: To obtain long-term embryogenic lines of

Das and Rahman


both genotypes i.e. Purbi and Bedana,proembryonal masses (PEMs) obtained in CIMwere cultured separately in a long-termmaintenance medium (LTMM) containing sameconstituent of salts as culture initiation medium.To develop LTMM, the effects of combinationsof three growth regulators, IASP, 4 μM; 2,4-D, 2µM; (1,2,3,-thiodiazol-5-yl)-N’-phenylurea (TDZ),0.2 μM were tested in a randomized completeblock experiment in a 2 × 2 × 2 factorial with fourreplications. All factors other than growthregulators were as described for CIM. Each blockincluded eight different treatments, in a differentpetri plates. Five inocula consisting of PEMs,approximately 2.0 mm in diameter were platedin each petri plate. After 60 days of incubationunder the culture conditions described previously,the experiment was evaluated. The efficiency ofeach treatment was determined by measuringdiameter of the calli at the surface where the cellshad been in contact with medium. Some culturesof both genotypes were kept in standard culturalconditions for maintenance of embryogenic linesfor long time (approx 2 years) and were subcultured in every two weeks interval and somecultures were transferred from LTMM to EDMMseparately of both cultivars for embryodevelopment, maturation, germination andregeneration into litchi plants. Sometimes totestify the competency of embryogenic lines forembryo development were cultured on EDMM,germinate and regenerated into litchi plants tostudy conversion efficiency. These embryogeniclines can be used in transformation experimentsto express agronomically important trait genesto develop genetically modified litchi plantswithout spending more time in regenerationsystem.

Embryo development and maturation: ThePEMs that proliferated in LTMM wereregenerated in an embryo development andmaturation medium (EDMM), which containedthe same minerals and organic nutrients as CIMbut altered growth regulators. EDMM included17μM IASP, 10μM α-naphthoxyacetic acid (NOA),1μM TDZ (Thidiazuron), and 1μM ascorbic acid

(ABA). In addition, the effect of three differentfactors in the medium were tested: 2.5 gl-1

activated charcoal (CH), 50 gl-1 polyethyleneglycol (PEG, MW 3,350 Sigma-Aldrich), and 20vs, 60 gl-1 sucrose. The plated PEMs wereincubated in a culture room maintained under 16-h day cycle at 25 ± 2 OC and light intensity of 60µmol m-2 s-1 (cool white fluorescent light). Theexperimental design was a randomized completeblock in a 2 × 2 × 2 factorial, with four replications.Each block contained eight different treatmentsand each treatment was plotted in a separatePetri dish. Five randomly selected inoculaconsisting of PEMs (ca.2.0 mm in diameter) wereplated in each petri plate. After 45 days ofincubation, the number of mature embryos ineach callus was counted.

Histological studies: For histological studies,the embryos at various stages of embryogenesiswere fixed in FAA for 24 h. Tissues weredehydrated by transferring embryos through anethanol-xylol series and then were infiltrated andembedded in paraffin and sectioned longitudinallyto 6 mm slices with a microtome. The sectionswere then stained with toluidine blue O for 10min, washed in water, dried at room temperature,stained with Lugol reagent for starch detection,and washed in water and dried again. The slideswere mounted in synthetic resin (Permount) andphotographs were taken with Nikon lightmicroscope.

Germination and conversion: The germinationof mature somatic embryos were accomplishedin the two Indian cultivars i.e. Purbi and Bedanaon MS medium containing MS salts, B5 (19)vitamins with 1 mgl-1 kinetin, 5mgl-1 gibberellicacid, 50 ml l-1 Coconut water, 3% (w/v) sucroseand 7.0 gl-1 agar. pH was adjusted to 5.6 prior toautoclaving. The germination tests wereperformed in a series of 100×15 mm disposablePetri dishes in replicated experiments (fourreplications). Thirty completely developedembryos were harvested from EDMM andtransferred to each plate. The plates wereincubated in the dark for 7 days and thentransferred to light in a culture room maintained



at the conditioned described above. After 14 daysunder light, germinated embryos were transferredto conversion medium (CM) containing MS (20)salts, vitamins, 20 gl-1 sucrose, 100 mgl-1 myo-inositol, 0.25 µM BAP, 0.1 µM NAA, agar 7.0gl-1 and pH was adjusted to 5.6 prior toautoclaving and dispensed into jam bottles. Thegerminated plantlets were allowed to grow in thismedium for 30 days and then transferred to MSmedium free of growth regulators. The plantletswith fully expanded four to five leaves establishedon this medium were further transferred to potsin soilrite. They were acclimatized andsuccessfully transferred to the greenhouse. Theacclimatized plants exhibited normal phenotypeand growth habit similar to the original plant.

Statistical analysis: The measurement ofembryogenic culture and the number of embryosproduced from all of the calli under differenttreatments were done under stereo microscopeand the data recorded as the mean of diameter(mm) as well as the mean number of somaticembryos in each experiment. The statisticalsignificance was calculated by one-way ANOVAfollowed by Tukey’s multiple comparison tests.All data analysis was performed using the GraphPad software (Graph Pad In Stat. Software Inc.San Diego, CA 92130, USA).

ResultsEmbryogenic culture establishment:Embryogenic cultures were successfully inducedfrom zygotic embryos in CIM. Since somaticembryos are repetitive in nature, calli were againdedifferentiated from these embryos, whenplaced in modified CIM (culture initiationmedium). The PEMs like structures wereobserved 45 days after initiation of culture, forPurbi and Bedana (78%, and 52% respectively).PEMs were slow growing, friable, white to dark,with nodular texture (Fig.1 A-D). Duringestablishment stage of in vitro culture both Indianlitchi cultivars i.e. Purbi and Bedana explantssecrete a large amount of polyphenolics whichupon oxidation with the help of polypheno--loxidase, turns brown color (melanin pigment),necroses and death of explants which checks

the in vitro culture i.e. proliferation of callus,somatic embryogenesis and regeneration of litchiplants. In Chenghe Yu et al. (1) (Chinese authorsworked on Chinese litchi) protocol browningproblem was not mentioned but with slightmodifications (control of browning, period ofcultures etc.) we succeeded to get somaticembryogenesis and regeneration of Indian litchis.It seems that both Chinese and Indian litchis aremore or less same.

Long term maintenance of embryogenicculture: CIM was efficiently used to induceembryogenesis in both the Indian litchi cultivarsbut the medium was inadequate to maintain longterm embryogenic cultures as the embryosturned dark and eventually died. Based onpreliminary work we lowered the concentrationof auxins (IASP and 2, 4-D) and tested thesetogether with TDZ. After a few weeks in variousLTMM, the cultures developed globular pro-embryogenic masses. An analysis of 60 days oldcultures showed that 2, 4-D alone was the mostimportant growth regulator for the maintenanceof long-term embryogenic cultures in bothcultivars. It was observed that 2, 4-D wasessential for proliferation but also preventedprecocious regeneration and germination thatcould lead to the loss of embryogeniccompetence (Fig. 1D). The interaction 2, 4-D ×TDZ was significantly negative (p >0.05) whencompared with treatments containing only 2, 4-D or 2, 4-D + IASP (p <0.001) (Table 1) forBedana. The embryogenic cultures grown onTDZ-supplemented media differentiated andsometimes lost embryogenic competence (Fig.1D). Many abnormal embryos developed in themedium, which germinated precociously yieldingaberrant plantlets that failed to survive. However,treatments which included 2, 4-D + IASP yieldedlarger embryogenic colonies in both the cultivarswith a better response in Purbi (Table 1) (21).

Embryo development and maturation: In thepresent experiments, several factors were testedto induce the development and maturation ofLitchi chinensis somatic embryos. The resultsshowed that activated charcoal had a significant

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(p <0.001) effect on development of embryos(Table 2). Proembryonic masses (Fig. 1E)regenerated somatic embryos (Fig. 1F) whentransferred to medium containing charcoal.

The effects of PEG or sucrose alone or incombination were less (p <0.01) significant inembryo development and maturation. However,a significant (p <0.001) interaction between PEGand charcoal was observed in both cultivars(Table 2).The best combination was 50 gl-1 PEG,

2.5 gl-1 activated CH and 30 gl-1 sucrose. Theseinteractions increased the yield of matureembryos in Purbi and Bedana cultivars to181.5±5.54 and 148.5±4.27 per inoculated PEMrespectively in comparison to yields obtained inthe treatment that included only charcoal (Table2). No significant effect of the interaction ofcharcoal × sucrose was observed in thisexperiment.

Histological studies: Histology of somaticembryo-producing regions confirmed that theinduction of the development process wasembryogenic and not organogenic in nature. Lightmicroscope observations of embryogenic massrevealed the presence of nodular structurescontaining cytoplasmic cells at the central region.Development of somatic embryos appeared toprogress through typical globular-, heart-, andtorpedo-shaped stage embryo development. Thefirst sign of embryogenesis was marked by theappearance of globular (Fig. 2A) structures theheart stage embryo (Fig. 2B-C), which wasbilaterally symmetrical and also showed a broadsuspensor like stalk. Some of the structures alsohad vascular tissue with unipolar meristemswhich ultimately developed cotyledonary somaticembryos (Fig. 2D). The dark stained meristematicarea was often surrounded by parenchymatoustissue. The studies of the embryogenic explantsshowed that they were not very rich in starchgranules but were rich in phenolic compoundsthat had accumulated primarily in a superficialzone between layers of cells of the peripheralregion and storage parenchyma.

Germination and conversion: Germination ofsomatic embryos is characterized by cotyledonexpansion and chlorophyll formation, followed byradicle and hypocotyls elongation. In the presentstudy, 65 % somatic embryos of Purbi obtainedfrom EDMM germinated well but only 32%converted into normal plants with roots, shootsand leaves whereas only 42% somatic embryosof Bedana germinated well and 17% convertedinto normal plantlets (Fig. 1F-J) in MS medium.Many of the somatic embryos of these cultivars

Table 1. Increase of ‘Purbi’ and ‘Bedana’embryogenic culture diameter (mm) after 60 daysof culture under different treatments, measured atsurface of the media where the cells had been incontact. Mean diameter of embryogenic culture offour independent experiments ± SE.

Treatment Basal Cross Section (mm) Meandiameter ± SE

Purbi Bedana

Control 2.97 ± 0.19 2.95 ± 0.11(without treatment)TDZ 3.05 ± 0.13 3.12 ± 0.16IASP 3.45 ± 0.25 2.92 ± 0.092,4-D 7.40 ± 0.36 6.25 ± 0.19TDZ + 2,4-D 8.22 ± 0.20 3.05 ± 0.18TDZ + IASP 3.25 ± 0.17 2.92 ± 0.082,4-D + IASP 9.42 ± 0.29 9.10 ± 0.492,4-D + TDZ +IASP 5.30 ± 0.18 4.27 ± 0.23

Table 2. Number of ‘Purbi’ and ‘Bedana’ matureembryos developed from 2.0-mm embryogeniccallus, after 40 days culture in EDMM under differenttreatments. Mean numbers of embryos of fourindependent experiment ± SE.

Treatment Number of Embryos

Purbi BedanaControl(without treatment) 19.50 ± 1.70 20.50 ± 1.04PEG 46.75 ± 1.93 39.50 ± 2.90CH 125.00 ± 2.64 77.50 ± 5.95Sucrose (S) 24.25 ± 2.78 20.00 ± 1.82PEG +CH 155.25 ± 5.18 139.75 ± 3.32PEG +S 73.00 ± 4.67 33.25 ± 2.42CH +S 61.75 ± 4.32 50.50 ± 4.57PEG +CH + S 181.50 ± 5.54 148.50 ± 4.27



Fig.2. Different stages of somatic embryogenesis (arrow) of litchi in longitudinal sections(A Globular embryodevelopment on the surface of explant (B) Early heart shaped embryo (C)Late heart shaped embryo (D)Cotyledonary stage somatic embryo.

Fig.1. Regeneration of plantlets from embryonic calli from 2.0 mm diameter of somatic embryos after 40-60days in EDMM (A) Immature litchi fruits from which zygotic embryos were excised (B-C) Proembryogeniccallus developed from zygotic embryo after 45 days of culture in CIM (D) Long-term embryogenic culturegrown in LTMM supplemented with 2,4-D (E-F) Viable embryogenic callus grown in LTMM supplementedwith TDZ and again culture on EDMM supplemented with charcoal for controlling of browning for thedevelopment of nodular embryogenic callus which later developed a large number of somatic embryoswhich were isolated from embryogenic callus. (G-H) Developing plantlet from germinating somatic embryo(J) In-vitro developed litchi plantlet (K) Field grown litchi plantlet.

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had roots and cotyledons but failed to convertand some were albinos. These normal plantletshave been moved to a greenhouse where theyhave been growing normally (Fig 1K).

DiscussionThe present study describes results

obtained for the maintenance of long termcultures for initiation and development of somaticembryos from zygotic embryos of two Indiancultivars of Litchi chinensis for which protocolswere developed earlier for regeneration (1). Sincedevelopment of embryogenic calli from zygoticembryo explants takes 9-12 months and it islabour intensive, so we have developed anefficient protocol for long term maintenance ofembryogenic calli from zygotic embryos whichtakes 3-months (unpublished data). Thus withthis protocol, without compromising theirphenotypic structure, we are able to reduce thetime needed to get embryos for these and abovementioned applications. Most of the successfulprotocols reported for litchi species, have beendeveloped for Chinese litchi species and forinterspecific hybrids. Successful plantregeneration in litchi has been reported lessfrequently and in only for a few cultivars (22). Inthis study we have observed somaticembryogenesis and methods for long termembryogenesis as well as high frequencyregeneration of normal plantlets in litchi. Theestablishment and maintenance of embryogeniccultures in litchi sp. is highly genotype dependentand thus far has been confined to a few speciesand their hybrids. The combination of plantgrowth regulators is essential for somatic embryoinduction, and that explants will not develop onmedium depleted of growth regulators. 2, 4-Dwas the main auxin giving the early results (23,24, 25). Kikert et al. (26) reported a low frequencyembryogenesis (0.1-3%) in anther cultures ofVitis lambruscana, after nearly one year fromthe initiation in various combinations of auxins(2,4-D and NOA) and cytokinin (BAP and CPPU).The higher frequency and precocity ofembryogenesis in CIM represent the synergisticeffect of combining several auxins. Similar results

were not observed if media were supplementedwith each of the phytohormone. These resultsmay be credited to the synergistic effect of IASPand 2, 4-D in inducing embryogenesis in litchi.The synergistic effect of these two auxins waspreviously reported by other authors in grape (27,21) and it was used the same combinations ofgrowth regulators to induce recallusing in maturesomatic embryos of litchi. IASP was alwayssuperior to free IAA. Most of the IAA in planttissues exists as conjugated or ‘bound’ IAA, thelatter form is relatively inactive and less sensitiveto catabolism or degradation and can be storedor transported in the plant (28, 29).Varioussynthetic conjugates of IAA have been found toevoke responses similar to those produced byIAA (29). Moreover, some IAA conjugates weremore active in inducing soybean callus growththan free IAA (30).

In transformation system, transformantsare more efficiently selected from highlysynchronized cultures of litchi because thesensitivity of litchi embryogenic tissues toselection agents varies in different stages ofembryo development. Synchronized long-termembryogenic cultures were obtained in thetreatments which included 2, 4-D alone or 2, 4-D+ TDZ or 2, 4-D + TDZ + IASP. In litchi, auxins,especially 2,4-D have been very effective forinducing somatic embryogenesis (1) it is not usedin embryogenic callus culture maintenance. Wehave observed that the embryogenic lines canbe maintained for long time (approx. 2 years),under frequent subculturing in LTMMsupplemented with 2,4-D with no loss ofembryogenic competence and embryodevelopment in litchi (21).

TDZ is among the most active cytokinin likesubstance highly effective in the induction ofsomatic embryogenesis on leaf callus of litchi (5).The negative interaction between 2, 4-D and TDZin Purbi and Bedana in litchi embyogenic culturesmay be due to the unsuitable ratio of these growthregulators for these cultivars.



Activated charcoal favours the growth anddevelopment of somatic embryos in litchi (21), itmay also be attributed to the establishment of adarkened environment, adsorption of inhibitorysubstances (phenolic compound), adsorption ofgrowth regulators and other organic compounds(31) secreted from the plant tissue during in-vitroculture, essential for long-term maintenanceof PEMs. However, in Myrciaria aureana,charcoal was found to be detrimental to theprocess (32).

The combinations of PEG and sucrose inthe medium have been more effective in thedevelopment, maturation and quality of somaticembryos (33 ) which also increase the depositionof storage proteins similar to zygotic embryos(34). Germination of somatic embryos ischaracterized by cotyledon expansion andchlorophyll formation, followed by radicle andhypocotyls elongation in suitable medium.Sometimes MS basal medium was found to beinhibitory for somatic embryos germination. Thiscould be due to a lower requirement for a nitrogensource and calcium ions for germination (22).Good quality of embryo production is importantfor increased rates of germination andconversion, which are limiting steps for a practicaluse of somatic embryogenesis of litchi (1). Withthe purpose of preserving the embryogeniccultures, different strategies have beendeveloped in litchi: recurrent secondaryembryogenesis without a dedifferentiated stage(35, 36), recurrent cycles of secondary embryo-genesis but alternating the differentiated anddedifferentiated stages (37, 15, 27, 38, 25) orthe long term maintenance of embryogenic callusculture (36, 39, 40, 41, 42, 12, 21, 43).

This protocol presented here constitutes avariation of the long term embryogenic callusculture, with low level of plant growth regulatorsto minimize the occurrence of somaclonalvariations and maintain the embryogenic culturesin a non differentiated state. Our results in litchicultivars were within the range of data reportedin other cultivars of grape also (36, 44, 42, 21,17, 38, 43).

ConclusionThis is the first report of successful long

term maintenance and recovery of plants fromembryogenic cultures of Litchi chinensis of twoselected Indian cultivars that could be used forcryopreservation. Proembryonic lines obtained inthis research are highly synchronized and eachmajor developmental stage (i.e. proembryo,mature embryo and germination) can becontrolled by varying the constituents of media.This control should also facilitate the recovery oftransformants efficiently.

AcknowledgementsWe, authors are grateful to the University

Grants Commission, Govt. of India, New Delhifor providing financial assistance. We are alsothankful to Dr. V.K. Tiwari, NIHFW, New Delhifor statistical analysis of various data.

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