Journal of Scientific & Industri al Research

Vol. 591July 2000, pp 531-540

Development of Clones and Somaclones Involving Tissue Culture, Mycorrhiza and Synthetic Seed Technology

Satyawati Sharma*, Suman Kashyap and Padma Vasudevan

Centre for Rural Develop ment & Appropriate Technology, Indian In stitute of Technology, New Delhi II 0 016, Ind ia

Under current sit uation of rapid ly increas ing population, wastelands, and unemployment generation, it is necessary to produce the quality biomass at faster rate for various purposes. i.e., food, ll bre, fuel , fertilizer, fodder, and agro-industries. In thi s regard , tissue culture has proved to be an efficient , eco- friendly, income generating, and technoeconomically viabl e techno logy which can be suitable for rural as well as semiu rban areas. The present review deals with the work done in producti on of clones , somaclones along with mycorrhi zation . and synthetic seed technology for reclaiming the wastelands (mainly salt affected areas). Synthetic seed production is an appropriate technology for rural areas as it does not require sophisticated laboratories. Fu rther, mycorrhization of mi cropropagated plantlets either involving tissue culture or synthetic seed technology hardens the saplings under fie ld conditions.

Introduction

Plant cell/ti ssue culture is rapidly developing technology which ho lds promise of restructuring agri cultural, horticultura l, and forestry practices. The technique immensely reduces the labour and space requirements for producing new va ri eties of desired

characteri sti cs and can a lso . markedly enhance propagation rates. Development of plant tissue cu lture is limited to improvement in techniques of protoplast , cell , ti ssue, and organ culture, followed by the success ach ieved in regenerating whole plan t from cultured plant materials. Plant tissue culture is presentl y of great interest to molecular biologists, plant breeders, and industri al ists. Tissue culture methods have been employed as important a ids to conventional methods of pl ant improvement. It is particul arl y useful for

multiplication of plants which are: slow growing; c ross-pollinated, and show wide vari ati ons in the progeny: male steril e lines; newly produced vari eties; for mul tiplication of virus free plants by meri stem cu lture. It has a lso been useq as a tool for the propagat ion of genetica ll y manipulated superior c lones, somac lones, in ex-situ conservation of va luab le germplasm, and synthesis of many important

*Author for correspondence

Fax : 91- 1 1-6R62037

secondary compounds. Clone propagation is a method by which identical plants of superior specimens can be obta ined within a short span of time. Work on media standard izati on and conditi ons for clonal propagation of plants have been undertaken in several ti ssue cu lture laboratories. The present paper reviews the work

conducted in the area of deve lopment of clones, sa lt tolerant somaclones, synthet ic seed technology, and micropropagation a long with mycorrhi zati on.

1 Development of Clones (Micropropagation)

Micropropagation is defined as "the true-to-type propagati on of a se lected genotype invo lvi ng regenerati on of plants from organs, tissues, ce lls or protoplasts using in vitro cu lture techniques".

Ball1

has successfully raised transp lantab le whole plants of Lupinus and Tropaeolum by cu lturing the ir shoot meristem (tip). The potential of this work was soon realised by Morel 2 for rapid propagation of orchids Cymbidium and Odontoglossum. The advantage of using thi s method was that about four million genetica ll y identical plants cou ld be obtained from a sing le bud. Murashige and Skoog' havt:' deve loped standard methods of propagation in vitro of spec ies ranging from ferns to foliage, fl ower, and fruit plants. Thi s new method of vegetative propagation has been exploited ex tensive ly in horti culture and the nursery industry for rapid clonal propagation of many

532 J SCI IN]) RES VOL 59 J ULY 2000

dicotyledons, monocotyledons, and gymnospermsworldover. The major advances in the masspropagation of woody plants have been made over thepast 10 y.

In India also, over the past three decades,considerable work has been done in the application oftissue culture technology to horticulture, sylviculture,and agriculture. Micropropagation of elite forest treegenera such as Santalum album, Eucalyptus, Tectonagrandis, and Dalbergia latifolia, even from tissuesisolated from mature, 100-y-old trees and from the oilpalm have been made at the Plant Bio-TechnologyDivision of Bhaba Atomic Research Centre (BARC),Bombay; Indian Institute of Science (IISc), Bangalore:National Chemical Laboratory (NCL), Pune; CentralPlantation Crop Research Institute (CPCRI),Kasaragod, Kerala, India. Significant contributions inthe use of tissue culture methodologies formorphogenetic studies on a wide variety of testsubjects was made by the Department of Botany,University of Delhi. The successful growth of ovary,ovule, and embryo parts in fruitset and seeddevelopment under ill vitro conditions has beenhighlighted, as response to exogenous hormones".

Since 1980, micropropagation of severalleguminous shrubby and timber trees has beenreceIVIng considerable attention. Also, clonalmultiplication of many ornamental plants like orchids,roses and chrysanthemum, fruit crops such as apples,peaches, pears, strawberries, plums, and forest treesnamely Leucaena leucoccpha!a, Prosopis cineraria,Dalbergia latifolia, Tectono grandis. Salix, and Fagushas been undertaken by various researchers.

Recently, callus and plantlet regeneration fromleaf explants in Bauhinia Sp.56, from shoot tips of Ficuselastica'; from shoot tips of cashew seedlings", fromhypocotyl and cotyledon explants from various treesand in vitro induction of tlowering and fruiting inMoms alba') was successfully performed. Variousprospects of in vitro genetic manipulations likeproduction of haploids, triploids, somaclonal variation,somatic hybridisation, and synthetic seeds of mulberrywere described by Chand et al.", High frequency budbreak and multiple shoots were induced in nodalexplants and apical shoots of various species ofmulberry on Murashige and Skoog's (MS) mediumsupplemented with 6-benzylamino purine (1.0 mg/l)along with gibberellic acid (0.3 to 0.4 mg/I)II.l'.

Mulberry plantlets were also "llCl,'"from callus cultures and inters].was observed!·1l5.

. '!v regenerated. ,"utoplast fusion

2 Development of Salt- tolerant Somaclones

It is well known that genetic variations occur inundifferentiated cells, isolated protoplasts, calli, tissuesand morphological traits of regenerated plants. Thecause or variation is mostly attributed to changes in thechromosome number and structure, Cytologicalhe+ rogeneity in cultures arises mainly due to suchf,l:lors as:

(a) The expression of chromosomal mosaicism orgenetic disorders in cells of the initial cxplants,and

(b) New irregularities brought ahout by cultureconditions.

These variants selected in tissue culture havebeen referred to as calliclones (from callus cultures)"or protoclones (from protoplast cultures'i'". Larkin andS f· 18 . dcowcro t comec a general term Somaclonalvariation for plant variants deri vcd from any form ofcell or tissue culture. The plant variants with saltresistant characteristics have been developed ill vitro toreclaim salt affected wastelands. The diverse variationcharacteristic of somacloncs highlights the fact thatsomaclonal variation may be an additional tool for cropimprovement rather than an interesting scientifich 1')·11 S I I .. ~p enomenon -. ornac ona vananons may be

heritable as established in wheat and hence useful ingenerating progenies showing desired characteristics I').

Plant tissue culture techniques have been used todevelop salt tolerant somaclones either by directlyexposing cells/calli/protoplasts/microsporc/anther orthe explants to single salt(s) or their mixture in one stepor gradually (stepwise) or indirectly by exposing toosmotic stress induced by mannitol or PEG(polyethylene glycol) or proline analogues:", Thepotential advantages of tissue culture techniques for theproduction of salt tolerant plants are:

(i) Evaluation and selection of large number ofgenotypes in a small space in laborato. J •

(ii) Reduction of time between generations.(iii) Control of environment and nutrient

conditions.(iv) Reduction of complications due to differences

in morphology and stages of development as

-------------------- .............--

SHARMA et a/.: DEVELOPM ENT OF CLONES & SOMACLONES 533

cells growmg tn culture are uniform in

development. (v) Generation of large vari ati ons with genotypes

may be there during culture. (vi) Selection of traits at cell level can be

evaluated in regenerated plants and the ir

progeny. (vii) Use of iso lated protoplast/cell/callus cultures

to study the phys io logical and biochemica l processes which regulate the sa lt stress

tolerance23.

The salt to lerant cell lines was first isolated from Capsicum annum by Dix and Street24

. Since then such cell lines have been isolated from more than 36 species of 28 genera be longing to 16 families. The variability

generated in plant cell s grown in culture can be examined for the des irable tra its at two levels25

:

(a) Isolation of variants wilhout cell selection - The callus is first regenerated into whole plants, which

are then screened by conventional methods. (b) Isolation of variants using cell selecfion- In thi s,

millions of cells , each representing a potent plant are cultured in a si ngle vessel exposed to conditions that retard or prevent the growth of normal ce ll s while favo uring the growth of

des ired variant ce ll s .

Callus-based select ion has been fou nd to be inefficient and uncertain as all the cells in the callus piece are not unifonnl y exposed to the se lect ive agent and this can result in stress avoidence due to cross­feed ing between the ce ll s in c lose contact with each other26

·27

. The ce ll suspension cultures have yielded over I 00-times more stable salt tolerance than callus pieces in Brassica juncea27

. But protocols for the establi shment and regeneration of cell suspension cultures are availab le on ly in a limited number of plant

spec1es.

Several somac lones of different crop spp. viz. sugarcane, potato, tomato , geraniu m, cereals, grasses, and lucerne, are developed in India but so far no useful vari ant of tree spec ies, particularly for sal ty areas has been developed . Salt tolerant cell lines of Pennisetum purpureum, an important pe renni al forage grass ; have been successfully regenerated into plants4 Salt toleran t cells in some of the plant species have been isolated by selecting the cells which ·accumulate higher leve ls of osmoregulatory compounds, e.g., proline quaternary ammonium compounds, po lyo ls, or by se lecting for

tole rance to osmotic stress component of salinity. At high salt concentrations ( 1.2-2 per cent) maize callus showed significant increase in tota l polyamine content, especia lly caused by rise in putrescine28

. In sugarbeet callus cultures, the salinity tole rance of the normal callus is accompanied by the accumulation of proli ne under hypersa line conditions (274 mM)29 Similar effects of salt (NaCI) with ten-fold inc rease in proline in alfaalfa cell lines30

, two to e ight-fold inc rease in ornithine decarboxy lase activity in roots and putresci ne contents in leaves of Vigna radiata was observed31

. On the contrary, the stress treatments of I 00 and 200 mM NaCI decreased the leve l of polyamines fo r the culti vated and wild tomato spec ies . Salt stress

increased the levels of diamine and po lyamine in varying degrees among nine ri ce cu lti vars in vest igated by Kri shnamurthy and Bhagwat" . Many amino acids

inc luding arginine, asparagine and serine were fo und to accumulate in both the sink and source ti ssues of Coleus blwnei during salinity stress34

. In spite of this , NaCI induced an inc rease in ethylene synthesis and 1-aminocyclopropane-1-carboxylic ac id (ACC) content in leaves of ri ce with exogenous putrescine app lication35

. Rice and Cicer ariefinum ca lli cou ld be grown under inhi bi tory levels of NaCI only in the presence of osmoregulants and there is accumulati on of proline implicated as an anti -st ress organic mo lecule in sa lt adapted callus. NaCI altered contents of nucle ic acids, protein, polyamines, and the activ ity of agmatine de iminase during germination and seedling growth of rice bes ides increas ing leaf morta lity prior to normal phase of senescence-'6.

37. In vitro selection of NaCI

tole rant ce ll cultures in two vari eties of 01yz.a satiwt was described by Paul and Ghosh 3~ . The organogenetic calli of Lycopersicon esculentum with tole rance of NaC I up to 20g/l and plantlets from callus grown on ISg/1 NaCI were obta ined and inorganic ion content in tolerant calli and somac lones was studi ed39

·40

3 Mycorrhization of Tissue Cultured Plantlets

T he accl imatisation of in vifro deri ved woody plantlets often leads to very high mortality rate due to des iccation and microbi a l in fection . Several efforts has been made to protect thi s by mycorrhizing the micropropagated plantlets both in vitro and in 1'i1•o conditi ons as both ecto- as well as endomycorrh iza have great potential in nutrient uptake, wate r absorption, biocontrol of pathogens, synthesis of growth hormones, and establishment of plantlets in

534 J SCI IND RES VOL 59 JULY 2000

adverse soil and climatic conditions'<". It was observedin situ and in vitro by Dixon et al.45 that introduction ofmycorrhizal fungi to unfavourable saline conditionsmay improve early plant survival and growth.

(a) Role of Endomycorrhiza

In several greenhouse experiments, inoculation ofmicropropagated woody plantlets with several spp. ofAM (Arbuscular mycorrhizai fungi increased survivalof the transplants from 80 to 100 per cent comparedwith uninoculated controls despite increasing biomass

ducti d . I 146-49 T' I dpro uction an nutnents eve . Issue cu ture

plantlets of two Paulownia species were inoculatedwith Glomus epigeium and the distribution of severalnutrient elements in these mycorrhizae was detectedwith X-ray microanalysis, spectra of which showedthat arbuscules, vesicles and hyphae contained more P,S, Mg, and Ca than the uninoculated plants". Influenceof AM on phosphate fertilizer efficiency in two tropicalacid soils planted with micropropagated oil palm(Elaeis guineensis Jacq.) showed that AM inoculationincreased the fertilizer utilization coefficient of plants2.7 to 5.6 - fold51

.

Inoculation with efficient AM fungi enhancedgrowth and mineral nutrition of five oil palm clonesand suppressed the variability between them52

.

Micropropagated banana plantlets growing in asterilized growing medium (1: 1 mixture of soil andsand, containing six ppm of NaHC03 extractable P)were inoculated with Glomus mosseae orG.monosporum or supplied with 33 ppm KH2P04,

fortnightly. Both AM inocula enhanced growth, P andN uptake and biomass production of host plants ascompared to the control. Moreover, Gimosseaeappeared to be more effective as a symbiont than G.monosporum, and this was probably due to a higherlevel of root cortex colonization and arbusculardevelopment'". Mycorrhizal plants developed a moreeconomical and efficient root system with goodnutrient absorptiorr". It was also observed by Fortunaet al.55 that growths of mycorrhizal inoculatedmicropropagated plantlets of apple and plum werecomparable to those fertilized with P. Hence,mycorrhizal inoculation can be used as abiotechnological tool to overcome blocked apicalgrowth and reduce P inputs to micropropagated fruittrees. In spite of this, mycorrhiza also appeared to alterthe carbohydrates status in stems and roots aspromoting total soluble sugar and starch contents in

micropropagated Pyrus and Prunus ' p'. 6. Inoculationwith AM fungi increased total shoot length, stemgrowth, and biomass of the plants with a welldeveloped mycorrhizal root system which alsoimproved top soil fertilit/7-60. Significant changes inroot system morphology, acclimatisation and growth ofrooted microcuttings was observed recently inmicropropagated plantlets due to mycorrhizal

bi . 61sym 1O<;IS .

Mycorrhization of plantlets in India was mainlydone by in vivo inoculation of AM fungi for properhardening of developed plants. Recently, evidence wasdocumented for the secretion of growth promotingmetabolites like indole acetic acid (IAA) and kinetin byAM fungi along with associative growth anddevelopment of micropropagated plantlets'", In vivoinoculation of endomycorrhizal spores in micro-propagated plantlets of Populus deltoides resulted inproper hardening at field level63. Similarly, inoculationof AM fungi on TC (tissue culture) plantlets ofjackfruit showed proper establishment of these in fieldconditions'", In a subsequent study on ex vitroinoculation of Leucaena leucocephala with Glomusfasciculatum, not only plantlets resulted in highergrowth rate of root/shoot, number of leaves, number ofnodules, and mycorrhizal colonisation but also only 20per cent of these survived in soils lacking AMfungus65,66.

Similarly, benefits of in vitro 'biotization' ofplant tissue cultures with microbial inoculantsincluding AM fungi was described recently byNowak67

. For the first time, the axenic AM inoculumof Glomus epigaeum was produced on Trifoliumpratense with the root organ culture techniques andapplied to the test tube plantlets of Paulowniaalbiphloea. Chemical analysis of plantlets showedinoculated leaves with more Nand P, same K, less C ascompared to uninoculated controls'". The work on theeffect of phosphorus sources on endomycorrhizalinfection of micropropagated bananas in vitro has alsobeen reported'".

Also, it has been shown by several workers thatthe mycorrhization of micropropagated plants at therooting phase in the sterile tube is much laborious thanthe mycorrhization at the acclimatisation phase anddoes not have additional advantage and inoculation atthe time of transplanting was much more effective instimulating growth".

SHARMA el a/.: DEVELOPMENT OF CLONES & SOMACLONES 535

Endomycorrhiza has a lso been reported to act as bi oprotector from diseases and pathogen attack, especially the nematode attack to micropropagated woody plants71

. Micropropagated pineapple plants of two varieties, Queen Tahiti and Smooth Cayene (c lone CYO) were inoculated at transplanting stage from axenic conditions with an arbuscular mycorrhiza l fungus to evaluate the importance o f endomycorrhiza

development for biological protection against Phytophthora cinn.amoni. Growth and minera l nutriti on of endomycorrhi za l plants were not affected by pathogen in comparison to reduction in growth of control , however, symbiotic functi oning wns reduced by the highest concentration of inoculum of

P.ci11namoni72.

(b) Role of Ectomycorrhiza

Ectomycorrhiza like endomycorrhiza a lso has a growth enhancement effect over micropropagated pl ants while inoculating in vitro/ in vivon 74

. The

advantage of expl oiting ectomycorrhiza in vitro is that not being obligate, it can be easil y cultured .

Various spec ies of ectomycorrhi za like Xerocomus badius, Paxillus invo!utus and Suiflus lacteus have good potential to store N, P, K, M g, Fe, Zn, AI, and Cd, and thus he lp in uptake and storage of macronutrients , ameliorate metal toxicity , and bioremidi ate the contaminated so ils75

-78

. Iso lates of Piso fithus tinctorius, Suilh1s luteus, and Laccaria sp. were found to be highly tolerant to salts like NaC I and Na2S04, in vitro conditions45

. P.tinctorius was found to be most effective in colonizing roots of Castanea sativa and mycorrhization of micropropagated plants

h d . . 79

increased survival and growt unng weanmg .

M . / 80 It was late r on observed by the artms et a . that physiological parameters of micropropagated Castanea sativa was greatl y influenced by P.tinctorius as it increased prote in content and photosynthetic rates, decreased the respiratory rates , overall mycorrhiza improved plants physiological status, thereby enhancing the acclimatization process . It was also shown by Simoneau et al. 81 while inoculating birch (Betula pendula) micropropagated plantlets with seven different isolates of the mycorrhizal fungus Paxilfus involutus that specific polypeptides were synthesised during ectomycorrhiza formation. Partial sequencing of

one clone have shown that it contained a portion of the gene for phenylalanine ammonialyase .

Eucalyptus marginata shoots and Pisolithus tinctorius were co-cultured to obtain ectomycorrhizal formation, in vitro. Successful combinations resulted in formation of a mantle followed by a Hartig net and epidennal cell elongation . Genotype, maturity, and

fungal specificity are key factors influenc ing successful ectomycorrhiza l formation of E. marginata by P.

. . . . 82 ttnctonus, tn vttro .

Pear shoot microcuttings inocul ated with I ml/1 Hebeloma sinapizans mycelium s lurry on MS medium supplemented with various concentration s of IBA showed three-fo ld enhancement of rooting percentage in H.sinapizans inoculated aux in free medium. Root

number/ plantlet was highest in inoculated media with or without IBA , but most roots were formed with IBA at 0.2 mg/1 83

.

The rooting pattern of Larix laricina hypocotyl cuttings under the influence of Laccaria bico for was assessed by Ste in and Fortin84 and compared with other plant growth regu lators. The results revealed that the hypocotyls cultured with the fungus in the auxin

supplemented media did not ex hibit any callus tissue formation and numerous root primordia were initi ated on the hypocotyl axis. Excised hypocotyls of Pinus halepensis were cultivated in vitro with/without Hebeloma hiemale on rooting medi a unamended or supplemented with IAA or tryptophan. H.hiemafe strongly stimulated rooting on med ia supplemented

. h h 85 w1t tryptop an · .

On the contrary , when three ectomycorrhizal fungi, Hebeloma crustuliniforme, Paxilfus inl'o futus, and Thelephora terrestris were inoculated in a peat/ve rmiculite mixture during the transition peri od of poplar plants from in vitro to glass house, survival of inoculated plants was found to be lower than that of uninoculated controls and lowest survival was observed in the presence of T. terrestris. But thi s fungus, however, gave the highest frequency of root infections with a significant increase in shoot he ight , doubling of shoot dry weight and an inc rease in N, P, and K contents86

. Micropropagated plantlets of chestnut were inoculated with Boletus edulis and B reticulatus mycorrhizal fungi and the ir antagoni stic effects towards Phytopthora (responsible fcir chestnut root rot) were studied by Chauvin and Salesses87

revealing anti pathogen activity of ectomycorrhi za.

536 J SCI IND RES VOL 59 JULY 2000

4 Synthetic Seed Technology

Synthetic or artificial seeds are the living seed­like structures derived from somatic embryoids/shoot axillary or apical buds in vitro after encapsulation by a hydrogel. The synthetic seeds provide a potential method to combine the advantages of clonal propagation with the low cost, and high volume capabilities of seed product ion . These also include the rapid and large scale multiplicati on, minimal labour, and low cost propagation of seed less, hybrid , and many vegetat ive ly propagated readily infected plants . Thi s technology is useful for multiplying transgenic plants. somatic and cytoplasmic hybrids, sterile and unstable genotypes and also for cryopreservation of desirable el ite genotypes. In additi on, artifical seeds can be directly delivered to the field , thus e liminating transplantati on and ti ssue hardening steps of micropropagation. The encapsulated embryos could a lso be packed with pestic ides, plant growth regu lators , fertili zers, nitrogen fixing bacteria, and even mi croscopic destroying worms88

·89

_

Redenbaugh et at. 'X> first di scovered that

hydrogels such as sodium alginate could be used to produce single embryo artificial seeds in alfaalfa and celery. The most usefu l encapsulation system has been fo und to be the dripping of 2 per cent sodium alginate from a separatory funnel into a I OOmM calcium nitrate so lut ion. Since then, artifi cial seed production and subsequent plant development of many important crop plants like brinjal, carrot, Brassica, lettuce, sandalwood, ri ce, etc ., has been demonstrated expl oiting somatic embryos, ax illary/adventitious buds, and shoot tips as in vitro propagules for

I t . 91•92 F I d . f . 'f' I encapsu a 1on . or t 1e pro uct10n o · an ti 1Ca1 seeds in tree spec ies, the recovery of plants (conversion) is crucial. Though difficulties in developing somatic embryogenesis systems in tree species are similar to those encountered in herbaceous species , tree spec ies ex hibit additional complexities. These are:

(i) T issue culture dependent variabilities are hi gh. (ii ) Genet ic variation may be hi gher in tree spec ies

than cu lti vated herbaceous spec ies. (i ii) Size of genome is quite large for many trees. (iv) Mature plant materi al is to be co llected from

fie ld .

In spite of these addi tional increase the potenti al difficulty repeatable somatic embryogenes is

variables which of developing

syste ms in tree

species, there has been tremendous progress in initiating somatic embryogenesis in trees9

' . Mang(fera indica was the first tree species for which the viab le artificial seeds have been produced94

. Afterwards synthetic seeds of mulberr/5

·96

, eucalyptus97 and '-) ) . '

banana have been sucessfull y produced. Following approach for genetic engineering of tree species with the help of artificial seed techolog/8 is recommended:

(i) Production of large scale e mbryogenic ti ssue from transformed (genetic ia ll y engineered) ce ll s or ti ssues.

(ii) Production of synchronous somatic e mbryos. (iii) Maturat ion of somatic e mbryos . (iv) Non-toxic encapsulation I coating as artificia l

seeds tran sformation and 'progeny' testing for low gene ti c and epigene ti c va ri ation, ex press ion of eng ineered trait.

(v) Cryogenic storage of potential superior lines. (v i) Greenhouse/nursery establi shment, growt h and

transplanting into fi e ld or plantin g th rough direct delivery.

There are ce11ain problems encountered 111

artific ial seed production like:

(a) Various c hemical and physical procedures are required to make embryo quiescent and viable for several months .

(b) Protec ti on of artifi c ia l seeds is required from desiccation.

(c) Convers ion from these seeds is often very low which may be due to improper development of embryo, or difficulti es to arrest growth or because of difficulties in development o f ' artific ial endosperm ' within the capsule.

(d) Protecti on of embryo against microorganisms is required by us ing fungicides and antibi oti cs. In late r stages , ho weve r, in oculation with mycorrhiza may be benefic ial 98

.

Conclusions

T he development of c lones (micropropagati on), somaclones, mycorrh izat ion of micropropagated plantlets and synthetic seed producti on are the effic ient technologies for fast product ion of many disease-free, superior plants with desi rable traits in :1 limited space fo r rec laiming wastelands. An integrated approach o f these technologies is requi red to overcome the increas ing de mand of food-supply and other va luable needs.

SHARMA eta/ .: DEVELOPMENT OF CLONES & SOMACLONES 537

In future, it is required to increase the development of clones/somaclones of important plant species and the screening, isolation, and multiplication of specific strains of mycorrhiza to protect these micropropagated plantlets under field conditions. Further, it is a lso desirable to enhance the output of somatic embryos per gram callus tissue, conversion frequency of embryos, preservation and protection of artificial seeds and encapsu lation of other propagules to develop new synthet ic seed coatings. The application of synthetic seeds should also be found in rapid delivery of tissue cu ltured developed clones and somaclones of different plant species.

These technologies wou ld not only play vital role in reclamation and afforestation of wastelands, but also cou ld help in meeting the gap between demand and supply of various biomass produce.

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