Ecology and Conservation of Biodiversity ore Ser P.Rovichondron cology and Conservation of Bodersty {© 2007 stonormarsm Sunderner Uriverty, Magnitude of Plant Molecular Biology and Tissue Culture Tools in Conservation of Biodiversity P.Ravichandran * Abstract Plant genetic resources ae the major biclogial bass ofthe world food security Inall meaas they support the ivliboods of evry lion planet, ‘earth. Biodiversity is the store house and act as a cushion against potentially dangerous envionmental changes and economic reforms. Such a buffer is facing treat due to man made and ecological disaster. Hence, conservation of Biodiversity is considered fundamental and provide priority ial sectors of lobal development Tratonal means fof germplasia storage and conservation of plant genetic rezourecs hae boon immenccly usefl and are not without drawbacks. Thus wting the biotechnological especially plant molecular biology and tissue culture appresches towards the improvement of in situ and ex sine ‘coneervation programmes are becoming vial. Ineratng biotechnology in plant conservation programs is a prerequisite to achieve success in ‘ustainability and to complement the existing technologies. In this context the conservation of plans trough in vitro propagation and inducing slow growth in some of the ezonomically important and threatened plans of southern Western Ghats is presented. During the Inst ten years of intensive research progeams, we are able to develop in vito protocol for rapid regeneration and establishment of plants in the Seld conditions for about thirty different species ofthis region. * ele cy nd Pit Bly Us Pon ae SoomIndia is major centre of origin and diversity of crop and medicinal plans. It holds an extraordinary significance among the tp gene rich ‘countries of the word relating tots abundantly ih land ace diversity in agricultural and horticultral crops and thee wild relatives. Inia possesses about 20, 000 species of higher plants and one third of it ‘bing endemic and 500 species are categorized to have medicinal value (Mandal, 1999; Arora, 1988). The southern Western Ghats is one of {he major repositories of endemic and medicinal plants, I harbors around 4000 species of higher plants of which 450 species belonging to 150 genera are endangered, The ri ist category inthis region is incteas- ing andthe valuable genetic reources ae bing lst ata rapid rate due to habitat destruction, environmental changes, natural calamities and ‘more reasonably through over exploitation. Tissue culture appreaches have been vital in the restablishment of endangered plant specie ‘This chaper highlights the integration of biotechnological programs into conservation of biodiversity as well asthe success achieved towards conservation of important plants trough micropropagation, Role of Biotechnology ‘The tools of modem biotechnology including molecular biology and tissue culture are being increasingly applied for plant diversity characterization and undoubtedly they have a major role in assisting plant conservation programmes. Howeve, ther valueis dependent upon ‘ensuring that biotechnological methods are targeted effectively and lized as complementary and enabling technologies. Most importantly, ‘they must be applied in the appropriate context. Biotechnology advancing so rapidly that it may be sometimes dificult for potential “cangrvaton’ users to asses the value and ole of new techniques and procedures within their own specific area. Ii important to recognize thatthe effective integration of bictchnolgy in consenation programs ‘equres muli- and interdsciplinary co-operation. Thus, present and ature conservation teams should comprise personnel from a broad spectrum of dicilines ‘There are four main areas of biotechnology, which can assist plant Gonsernvation programmes, 1) Molecular marker technology, 2) Molecular diagnostics, 3) Tissue culture (im vitro ‘echnologies) and 4) Cryopreservation, In addition, “information technology” (7) will have increasingly important role in facilitating ‘conservation programmes and the interface between IT and ‘biotechnology provides considerable potential for many aspets of plant gendtie recource management [Molecular markers and diagnostics For economically important plant specie, itis also essential to consider the relationship between conservation and utilization and to recognize ‘that biotechnology can enable sustainably programmes (Benson, 1999) “The luidation of population structures and gene distribution pattems within ecosystems provides information, which ean be wed to support Jn sine conservation programmes. Contrasting examples of techniques include the assessment of restriction fragment length polymorphisms (RFLPs) which permit the (PCR) detection of specific markers gees land polymerase chin renction-based marker technologis_wsed in a fociation with randomly amplified polymorphic DNA analysis (RAPD). ‘Tissue culture programs ‘sustenance of ie on the Earth, Development of suitable methods and formulation of ideological strategies to avoid the depletion of natural fesourees becomes vital Since the dawn of civilization man bas lara land executed various cultaral practices at Agriculture and Horticulture. ‘One such method developed with more scientific knowledge inthe ast five decades is tissue culture. A techaique whichis now wed for in ‘iro malipiaton of plants within short period in controle conto "Tissue culture is curently alzo employed to conserve all wil plant genetic resources particularly the rare, endangered and economically {mmpertant plants for posterity to make them available is noaded. The need for germplasm collection and conservation in the context of rapidly diminishing natural and culated species is warranted and appropriate “Tissue culture (on vitro) technologies hve hada major impact onthert Fat ‘exsita conservation of plant genetic resources and importantly, disease indexed in vitro maintained germplasm provides an excelent meas of ‘mediating international germplasm exchange, Micropropagation, using somatic embryo and shoot ip culture techniques asst many crop plant improvement programmes and increasingly these methods are being ‘sed for the conservation of endangered plant species. Crop plants, which are vegetatively propagated present particular conservation problems, as ther seeds are not available for banking. Whit fed {gencbanks provide important conservation options, germplasm ‘maintained in this manner can be at risk from pathogen attack and climatic damage. For vegetatively propagated species, in vitro conservation using tissue culture methods is the only reliable and long-term mean of preservation Storage inthe ative rowing sate or under reduced (slow) growth provides cost effective and medium-term ‘conservation options. Most major, international germplasm centers use in vitro conservation as their method of choice for vegetatively propagated crops. With the. development of tissue culture techniques the problem has been solved to some extent. There are two methods of Jong term storage of tisuceutures- the fist is by slowing down growth and th second by arresting growth by string the sues at very low temperatures. Cryopreservation ‘Maintenance of plant germplasm in the active or slow growth state provides 2 medium-term storage option; however the long-term conservation of vio -drived plant germplasm increasingly achieved ‘using conservation in Tiqud nitrogen. Cry conservation sthus applied to plant germplasm, which cannot conserve using traditional see banking techniques, ad /or to vegetatively propagated germplasm. In 1980, ‘Withers and Williams high lighted the problems associated with “dificult to store and seed recalcitrant germplasm’. Itis encouraging to note that during the Ist decade or mere, major advances have been rade inthe succesful application of ryepreservaton methods to once termed. “difficult” germplasm types (Benson, 1999). Particularly significant advances have been in the cryopreservation of the recalcitrant seeds (and excised embryos) derived from tropical agroforesty and planation crops and rain forest tree species. ‘Ravances in biotechnology Rave only been equaled by the activity of ‘the information science and technology’ sector. The ‘Infrmation Teeh- nology (IT) revoton’ i indeed rapidly changing the way and means in ‘which conservation scientists perform their research and implement their conservation strategies. On a practical basis, IT docs, and continue o assis all aspects of documentation associated with genetic resource transfer and management, genome mapping, DNA data basing and gene bank inventories. However, inthe future it will be important to enhance and consolidate the enabling role of IT in international training and technology transfer. Distance leaning and slesronie networking specifically designed for and targeted at plant conservation programmes will promote the expediency of concerted interational oneervation activities, ‘Conservation Biotechnology and the Sustainable Utilization of Plant Genetic Resources Secondary metabolites are organic molecules synthesized and stored in ‘plans in relatively low quantities. They may have no obvious role in ‘growth and development ofplant but have significant importance in self ‘defence. The natural products have enormous potential a meticinal, ‘utracetical and commercial valve. These include erpenes- pigments, ‘zsentil oils, steroids, rubber, phenolic compounds ~ coumarins, flavonoid, lignin, and tami, glycosides saponins, cardiac pcos, cyanogenic glycosides and alkaloids. All these compounds can alternatively be produced abundandly through cell culture without exploiting the natural resource. ‘The production of natural products (secondary metabolites) from the cll or teste cultures has log been a goal of plant biotechnology. I is {generally agreed that the potential for the syathesis of high value ‘natural produits fom plant cell cultures i tremendous. A great dal of effort has gone into the selection of high yielding cel Tins, designing culkure systems and formulation of culture and process systems. Two ‘major systems have been established forthe production of secondary maabolits:) through mas cell caltrei by immobilization of wile cls, ‘Mass plant cell cultures contribute new ways to the commercial synthesis of natural products: i) a8 a new production method forMei ‘Stablished products, frinstance morphine quinine and digorn if) asa ‘outeof synthesis to novel product from plants dificult to grow; i) as source of novel chemicals in thir own right; and iv) of increasing ‘importance as sources of enzymes and biotransformation systems cither on thir own o in combination with chemical synthesis. Plant cll _uspencion cultures involve growth of single or eellagregats in guid ‘medium, Cultured cells are made to synthesize useful metabolites. ‘Synthesis and accumulation processes are induced by altering media ‘compasitionand feeding of precursors and elicitors. Induction of changes in the shikimic acid pathway plays cenal role in symtesis of useful ‘Pharmaceutical. Enhanced production by cll immobilization and biotansformation of new compounds are potential areas of this research. Immeilization of “whole celle forthe production af secondary metabolites i a technolony’ nly recently developed. A selected line of cells for desired product synthesis i entrappe using range of gels. The most commonly used seis the sodium alginate. The entrapped cells can be cultured in bioreactors for easy separation of products. It azo provides an extended period of product recovery and increase stability of the caalyst. The bioeyntctic eapacity of immebilize plan cell enhanced by biotransformation, de novo synthesis and precursor feedings “The carl study ofbitransformation and using immebilzd plant cells was the hydroxylation of digitoxin to digoxin by Digialisfanata ‘entrapped in alginate. Since then a wide range of products were ‘obtained by simple biotransformation including L-DOPA in Mucuna ‘pruvens cells entrapped in alginate and transformation of codcionone to codeine in Papaver somniferum cells immobilized in alginate. Addition of precursors to immobilized cells has ao increased product “syutess. The supply of precursrs of indole alkaloids suchas typtamine and secologain to immobilized cells of Catharanihus roseusineseased the level of ajmalicine. Such sth advantage of ell and tissue culture forthe synthesis of new and high quality chemicals Alkaloids and terpenoids have extensively been produced in vitro conditions. Most of alkaloid compounds are pharmaceutical leas. Localization of specialized cells responsible for synthesis and accumulation of metabolites in plant tissues help extensively to choose ihe ell ype for culture. Altering the gonatc make up ofthe cls to go for enhanced production and quality drugs are the theme focus ofthis field. Hairy toot cultures and bioreactor production of secondary me- tabolites have revue in remarkable yields. Many prospective drues including as taxol, ajmalicine, atropine codeine, dopamine, dgitoxi, ‘morphine are being obtained enormously from in vil call cltres and thereby coneerving natura resource. 11995, the popular, UK-based plant conservation journal, Plant Talk, produced a communication entitled “Yew in the fight against cancer: ‘staiabilty or pillage?” The article refers, of course, to the use of Tras species for the production of the secondary metabolite taxol, ‘which is used to produce a potent anti-cancer drug, Whilst synthesis of the secondary product has been reported, and indeed, the drug has been launched in the US, the article presents some intresting facts, such that takes approximately ten Pacific Yew trees to yiekd enough bake forthe 2g of taxol required to teat single cancer patient. The lnk ‘between plant conservation and sustainable uilization (as opposed to exploitation is inded of major importance ‘Biotechnology can directly or indrectly enable conservation strategies, yet at the same time allow economically significant species tobe both tlie and protected. This sa major issue for those global ares, rich in biodiversity and for which there isan urgent need for populations to realize the esonmie potential of ther rch biological resourees and yt, at the same time preserve them for future generations. Prospects ‘Biotechnology snow integrated in all aspects of plant germplasm char- acterization, acquisition, conservation, exchange, and genetic resource ‘management. Future prospects are highly encouraging in terms ofthe evelopment and application of ew techniques and protocols within the context of gesmplasm conservation. The sustainable utilization of plant diversity can be greatly assisted by the application of direct and indret biotechnological procedures. Future prospects and necds must target certain key areas including; the development of appropriate structures for eryopreserved genebanks, the use of in vitro methods {forthe safe transfer of disease free germplasm, and the aplication ofi tapas cP [genetic marker technologies for atonaizing germplasm procurement and gene banking. As in vitro and molecular approaches to plant conservation become more and more important to validate routine ‘operational prtocols within and between gencbanks and repsitores. ‘This must be considered on an international basis and the provision of| ctworking and training infrastructures, withthe aid of T, will asst by ‘enabling ost effective training, and collaborative communications. ‘Whilst considerable progress has been made in the application of biotechnology to plant conservation there still emains the requirement to perform fundamental research, Seed recalitranc, tissue culture recalitrance,Somaclnal variation and cryopreservation injury ean be problematic for certain species. Similarly, whilst there has been considerable success in the use of molecular techniques our current Imowlege of the molecular biology of many groups of plans (eg. temperate woody perennial tropical ain forest tees) is stil limited Unlike many biotechnological ‘applications", conservation biotechnology programmes must be considered with a long-term perspective. Cryopreservel and in vito gencbanks, once reted, mist be maintained in perpetuity. Within an international context there is thus a need for individual governments and regional and global networks t have a commitment to provide sustainable and long-term funding. To date, many advances in plant conservation biotechnology hhave hada short-term remit o solv a particular conservation problem ‘or develop a exiain procedure INVITRO PROPAGATION AND SLOW GROWTH ‘The science of plant cel tissue and organ culture was conecived and counciatod by a German botanist, Haberlandt in 1902. He visualized the ‘dea of growing plant cells in artificial mai inthe hope of rejuvenating ‘quiescent cell and wigering it into division, growth, fo form a issue ‘and eventually, egeneate a whole new lant This is a function ofthe “totipotency” of plant cells, that is, thei ability to regenerate a ‘complete plan from a part. Each living cell has the genetic potential 10 ‘bring about differentiation of an ene plant, when place inthe right vironment of mineral nutrients, organic metabolites and hormones. Foe micropropagation, starting materials (propaguls) can be obtained ‘om picses of differentiated tissue of the leaf, from meristematic tissues such as vascular cambium, root and shoot ips, lateral buds and ‘young flower pats, or from mature pith and storage issue in stem. ‘Such tte pices are called explants. Plant cll, suc and organ culture is fundamental to most aspects of bioteshnology. The intrinsic property of excised shoots and roots to ‘regenerate a whole plant has been recognized for several decades. Applicaton of tse culture inclde Plant propagation i) genmplasm maintenance and storage, whch serucial for retaining the ene pools ‘of plant, i) the production of commercially useful chemicals and somatic hybridization and, genetic engineering ‘A now formation of roots and shoots is central to vegetative ‘Propagation by ctting. Tissue culture techniques considerably enkance ‘his potential by providing nutritional and hormonal regimes under aseptic conditions to very small segments of plants. Plant propagation by this method is called micropropagation because miniature shoots oF plantlets are initially derived. Micropropagation has become an important alternative to conventional vegetative propagation. Micropropagation is alo defined asthe tru to type propagation of a selected genotype in vitro. Micropropagation has two primary uses i) rapid clonal propagation and i) establishment, maintenance and istzibtion of pathogen fre clones. The principles and importance of tisue caltre has been extensively reviewed by George and Shetngton (1984), Debergh and Zimmermann (1991), Rice et al, (1992) and Narayanaswamy (1994), Pathways of Regeneration “Tissue culture has thre major pathways forthe regeneration of whole plants frm excised plant parts: i) regeneration of shoots from existing meristems; i) regeneration from adventitious meristem and ii) ‘regeneration though somatic embryogenesis. For some plans all the pathways may be appropriate, wheres inher plants only, one or #0 ‘may be possible given the curent level of understanding. Regeneration from existing meristems is achieved by culturing a12. Mag i ‘meristem containing shoot ip or axilary bud ora nodal bud Fig 2, 4, 10), Ona medium containing eytokinn, these explants proliferate into a cluster of axillary shoots due tothe loss of apical dominance in the original tissue. Shoots formed may be separated and subcultured onto ficsh medium fo farther proliferation of axillary shoots, This response has been demonstrated for a wide range of medicinal, ormamental and forest tee plants (Debergh and Zimmermann, 1991). The level and choice of cytokinin has tobe determined for each species, in order to obtain an aceptable rate of shoot multiplication, Regeneration of adventitious meristems involve the inition of new meristems either directly (Fig. 7) or after callus formation from explants of mature plant organs such 25 leaves, stems and r00s. Initiation of such meristems is controled by the balance of auxin and cytokinin inthe culture medium. Generally shoots are diferentiated on high cytokinin medium and roots on high auxin oF eytokinin free medium, Regeneration of plants through somatic embryogencsis (Fig. 3 & 5) has ‘een considered asthe most promising technique for rapid multpica tion of plants, Somatic embryogenesis, a process analogous to zygote embryogencsis, isthe development of embryos from one or more ‘somatic cel, begining wit the development ofa polarised structure possessing an embryonic apex and suspensor region. Somatic embryo- ‘genesis in both monocots and dicots has been well documented (Raghavan, 1986, Gray and Purhit, 1991). Somatic embryos are in- duced directly on explants such as orchard grass (an ornamental erase cultivated in higher elevations) leaf pieces and coffee laf discs. They «an aso be obtained through callus formation as cell in iquidsuspen- sion culture also yield somatic embryos suchas carrot Somatic embryos if they are produced in large numbers can be converted into artificial seeds, which may be stored and planted like ‘nomal eed. These somatic embryos are encapsulated with net chemi cals such as sodium alginate and polyacrylamide gel. Thus Somatic embryos are finding importance in synthetic seed technology. Redenbaugh et al. (1988) have described the various principles involved in the production of symthetic seeds. Synthetic seeds are already inthe market fr a numberof vegetable crops and oer plants in the developed count. ‘Stages of Micropropagation Stage 0 Preparation and pretreatment ofthe plant Stage 1 Initiation ofthe explant Stage? Muliplication of issue Stage 3 Regeneration of whole plans Stage 4 Hardening for subsequent fed planting Any explant or starting piece of plant material has to undergo the above ‘mentioned stages for rapid multiplication in vitro. For an easy “understanding a procedure followed in the case of shoot tp explant is described ‘Stage 0: Preparation and sterilization ofthe explant ‘Shoot ip explants of ess than | cm in length are sterilized by repeated ‘washing in tap water mixed with detergent solution. They are made ‘germ-free by dipping into alcohol or by treating them in 0.05% ‘commercial bleach like vaz(Soium hypochlorite). Aer the treatment ‘explants are thoroughly washed in sterile distilled water and then transferred aseptically into culture vials containing sterile medium. ‘Stage 1: Inidation of explant ‘The ination ofthe explant is most universally achieved in Murashige and Skoog medium (MS) which mainly consists of all major and minor nutrients, aminoacids and vitamins. ‘The metium is made semisolid by dissolving and boiling agar (0.8 - 1.5%), The medium is aso enriched wih plant growth regulators such as auxin and cytokinin. For shoct tip cultures, a higher level of etokinin such a benzyl aminopurinekinesin and lower level (0.1 -§ mg) oF 20 level of auxin would be necessary to initiate growth. The establishment of culture is also affected by factors such as the season when the ‘explants are taken. Spring oa period after summer is often found tobe the best time to take shoo tip as they possess considerable vigour and les infection,‘Stage 2: Multiplication of the ricsue ‘The mukiplcation of shoot tip explants is crucial in the propagation of sy spss comma epson ante mrp ats of ‘multiplication one requires. Single shoo tip multiply into many shots ‘du tthe loss of apical dominance and rapid multiplication ca: be achieved by altering the level of etoknin. Stage’ : Regeneration of whole plant ‘The regeneration of whole plants for many species which are propagated through tissue culture normally means the production of ‘oo, as axillary shoot proliferation remains the most common and ‘elable method for rapid malilication, "heaton of en ya in sae iby the rnin el i whe sie feta fon meting tuum, epecily idle utc aid (HA) (228 my). Th treat uy be lly non wih teed ds eg ofthe mean alten MS mdm) Shs develo ih og sunt so or teed he Sg Stage: Hardening / Acclimation for subsequent field planting Plan rit condo ad te lel eircoment inser way Pape he met ene he van (oe Sty en tt nl de ‘unter vo ras, Ape repatriation ped ‘ete aco onl tl of he eb pa inv The resin body te ane fr ost pera, Ps in thou are sem ah vine ot hgh on, cee ‘st chase ogg fone wl outa, The ate hii apt ih Ape wh te tel hw ene ay ‘ial oar cela te ef aos ev ‘phosphate may be beneficial, ie ‘The plants are gradually weaned by reducing the humidity, The stomatal response of tissue cultured plans is defective and the wax layer isalso drastically reduced on plants maintained in vitro, therefore transpiration losses willbe dramatically high therby reducing the chances of survival. It has been established tht a reduction of the relative . eee ee ee eee oy nadia 0 25 ‘Dumidity to 80% is sufficient to cable te formation of funcional ‘stomata and a sufficiently high wax covering to reduce water loss 10 ‘lose to the value for seeding material. Ths reduction in relative Inumadity is most readily achieved in vitro by loosening the vessel ‘locure and/or cooling the base of the vessel to 2-3°C below the air temperature. Plants (eat in this way and gradually weaned in a ‘relatively high humidity with shading havea far higher survival rate, GERMPLASM STORAGE, “The last seven years of accomplishments in tissue culture at our lab is compile here and presented in a nutshell. Micropropagation has been Suceessflly achieved either through direct morphogencss or indirect ‘moephogensis for abou thirty species of angiosperms inluding Some ‘rates for apd multiplication and proper establishment inthe fed and wild (Table 1). The procedures developed can also be utilized for ‘Conservation by inducing medium term-slow growth fr al these plant Species. Aminimam of 25 plantlets were caine from a single explant ‘bypassing the calls stage and thus achieve clonal propagation. Plants like Wendlandia, Curculigo, Aegle marmelos, Amorphophallus “ylation, Eryngium fotidum (Andaman coriander), Neem, Janakia, ‘Decaleps, Bacopa coud be maintained for years together by inducing slow growth. Slow growth was induced by altering the medium ‘equstes and hormonal balance, particularly the levels of anxin and fytokinin. Cetain plants such as Neem, Manisuris, Wendlandia. ‘Bacopa, and Janaki could be maintained even without any hormonal supplements in the culture and maintenance media for 3 longer period, hence achieved in vitro germplasm storage Plant breeders have contributed to the improvement of erops by combining desirable genes into a cultivar. In many cases the desired traits have been obtained by crossing with wild plants. The farmer's ‘contribution foo cannot be underestimated, as over the years they have Selected varieties suitable to thei locales. But bot these methods are endangered because ofthe erosion of the Biological diversity. Today ‘Sestuction of forests and other natural habitats is resulting in an alarm ing loss of plant spetes, I is expected that atthe resent rate, one quarter (about 60,000) of pant species could be lost by the mide of ‘he next century. Of the cutvars, a huge number has been lst due1 map the introdotion of only a few high yeMling varieties, We ae not only losing eutvars, but along with it we are losing the genes which might ‘one day come in handy to introduce a particular trait into a crop ‘specially when with the advent of biotechnology we can overcome barriers between incompatible species. We are also lesing a potential source of yet undiscovered food plants and those that provide other important necessities including medicines and biochemical. Wileitmay not be possible to save all the species and cuivars atleast a large number can be saved for the future generations through storage in ‘germplasm banks or gene banks as they ate commonly known. ‘The easiest and most economical way to save germplasm is ofcourse to save sods of known species and varieties under canditios in which their chance of remaining viable are high. Sead banking, however, has its problems. Some crop plants donot produce viable seeds. Some seeds are recalitran; these are usually the large and succulent sed, which Tack dormancy, and cannot withstand low temperatures or dey dation. Other seeds deteriorate rapidly because they are infected by pathogens. Some varieties ae heterazygous, and donot produce plants ‘with identical tats every time, Lastly one cannot wse sed banks for storage of vegetatively propagated plant. Ginger, onion, garlic and potato ‘can no doubt be stored, but not for long periods. Besides they are rather bully materials and would require a great dal of space. Visionary and sustainable funding policies, organized in concerted action by individual governments and appropriate international ‘xganizations will be essential to enable the next phase of conservation. Without such support it will not be possible to capitalize om the achievements to date ard use them to implement long-term, and safe, plant diversity conservation programmes. ‘Table 1. Micropropagation of Rare and Econ: Important Plants ‘WHCS [FAMILY [SIATUSUSE [peo ea [REPRINTS [scene sen, [Nant [Paina lose Riese | edn! | sedingenz,|Sérkaner Stoop {ais [abeveeL Burn | inese Ma —|Shotiandia| Wai es aang ‘si ote ne | Nae, erm, Jonamene_| eres | [aca ise [Merl —| Noa Eno, ancandan seeding ops, | 95 Stott “moons hia doce |RET pat | ome nits |Mio Exc anit | la. Bam. | opi] Nia —| Needs |e ctr po. \Gonahinipan| Caicos [Meinl [Neda | Veni, Dyepeig | sng oes. | 200, Stoop [Limca (GisirbaaSw [Rasoe [Feirend [Nedved Rainn Medal os (Geraipootis|Rpaiince| i — |S, ang ‘cam. Met, | tootip | Rovdannal ano lit pensar |Cpemane [Neca | ace, [Patina ot Maisie [Rime [Beara [amt [i [icon — [Rss] Si [vei api Wd | esis | rss | fz [danomell. | Soene Meson Auer Pot atin x [| ecephonlod apse [Ram | Shorey | Marel Iwan Ein, | Noe, lao Nes _| iattga taunts 122 aad 57] Sania [Simms | Maal | Ne aL 5 seam [ose Pot Bene Is ania eh ar rene loc Fi] Wns | Repel Na Ger ogee Sant fv | Bacon ao eno mm c REFERENCES: {a ‘Agalya Devi, N. 2004. Tissve culture and ccological adaptational oat Stodics on Decolepic hamilton Wish and Arn. ~ an Nex angered aromatic metic plat. MSs. Thesis, SPK Cente for Environmental Sciences, Manonmaniam Sundaram Universi Alwaraich Vn esas Arora, RK. 1988 Te Inia gen centre pirtesand prospects for eC ie celeton, I: Pads, RS. Aor, RK and Chandel, LPS P| ir mr (Eis, Plant Geni Resoures nan ppc, 49-516, : = New Deh: NBPGR. Names Beatie Valris, A Chandrasekaran, Ravichandran. 1953 lio Tegencraton of Manus murs. ~ An endef. FE ova. 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Hams ~ by mo- lecular markers and laf anatomy. M.Sc. Thesis, SPK Cen- ‘ae for Environmental Sciences, Manonmaniam Sundaranar ‘University, AlwarkarichFig. 1. Direct shoot multiplication from axillary buds of Aegle. sp.Fig.2, Direct multiplication of shoots from embryonie axis in Fig 4, Shoot multiplication and whole plant regeneration in Eryngium norphophallus sp. foetdhun (Andaman coriander) Fig. 3, Diflerent stages of somatic embryos obtained from Datura sp. Fig. 5-6. Somatic embryos ftom immature embryos and cotyledons of neem,\dvenitious shoots from hypocotyl segments in neem Fig. . Direct shoot multiplication from axillary buds of Aegle sp.