Applications of plant tissue and cell culture in the study of physiology of parasitism

A P P L I C A T I O N S O F P L A N T T I S S U E A N D C E L L C U L T U R E I N T H E S T U D Y O F

P H Y S I O L O G Y O F P A R A S I T I S M

BY RAMESH M/d-n~sltW/dl (Departme;It of Batca~y, U.ffversity of Delhi, Delhi-7)

Ree=i.'el May 23, 1968

ABSTRACT

The technique of growing plant tissues and cells on nutrient media under conditions of controlled environment has greatly enlarged the scope of experimental investigations. In phytopathology, application of this technique has given an insight into the nature of abnormal growth, factors affecting penetration, infection and multiplication of pathogens, the Weapons of attack of the pathogen, and the morpho- genetic potential of the diseased cell. The paper reviews recent applica- txons of the techmque in studies on parasitism and points some future possibilities.

INTRODUCTION

IN 1902 the German botanist Haberlandt suggested culturing isolated cells of green plants. This, he believed, should tell what a cell is capablz o" doing when freed from the influence to which "t is subjected within the

multicellular organism. Toward this obj:ctive, firstly organs, then tissues and later free cells have been grown in vitro. These three classes of culture have been commonly, although incorrectly, termed "Tissue Culture". Only in the past few years single somatic cells of some green plants have been induced to develop into entire individuals and eventually produce flowers and fruits (Vasil and Hilderbrandt, 1965; Steward et al., 1966). This has proved that a living cell has an inherent potentiality to produce a plant. That is a living cell is totipotent. Thus the objective with which plant tissue culture was initiated has been realized and tissue culture has now become a remarkably useful tool in experimental studies. In this paper the potential applications of this m~thodology in understanding the charac- teristics of pathological growth, the processes of infection by plant pathogens, their multiplication and the response of host o infection are discussed, A recent review has appeared on this subject (Braun and Lipetz, 1966). 152

Applications of Plant Tissue and Cell Culture 153

The advantage of the in vitro technique is the aseptic condition under which tissues and cells are grown. The recent work on auxin metabolism' demonstrates that results of investigations on plants grown under non- sterile conditions may be misleading. Libbert and co-workers (1966)have shown that the major quantity of indole-3-acetic acid (IAA) extracted from plant is not a product of the plant itself but of the epiphytic bacteria. Further, the ability of plants or crude enzymes prepared therefrom, to convert tryptophan to IAA is mainly the action of epiphytic bacteria. Works with aseptic plants have shown that IAA is largely synthesized from some source other than tryptophan (Libbert et al., 1966, 1967; Thimann et aL, 1967), probably indole-3-glycerol phosphate (Libbert et al., 1966). Therefore, the biosynthesis of IAA from tryptophan in plants, as described in texts of plant physiology, is questionable. Thus plant tissue cultures should find wider application in studies on metabolism.

With the discovery of vitamins and plant growth regulators it has become possible to grow some plant tissues on chemically defined media. This makes possible to control the chemical (nutritional) besides the physical environment for plant growth. Thus, the metabolic and structural changes brought about in cells by the manipulation of these factors can be followed. The effect of interacting factors on cell growth can be easily measured in terms of increase in weight. Comparative studies on nutritional requirements for the growth of different organs and tissues in culture can reveal differences in their biosynthetic capacities.

When agitated in a liquid medium plant parts and callus tissues derived from them yield a suspension of cell aggregates and free cells. Such a suspension can be serially propagated by pipetting aliquots into fresh medium. This bacteriological approach towards cell propagation offe s numerous advantages and opens new lines of investigations. Colonies of single cell origin, called clones, may be established. In this direction the first successful attempt with plant cells was by Muir et al. (1954) at Wisconsin (Plate IV, Fig. 1). The " Microculture Method " of Jones et al. (1960) (Text-Fig. 1) permits continuous observation on single cells as they divide and form colonies. Such techniques have been responsible for pro- viding unequivocal evidence of cell totipotency (Vasil and Hildebrandt, 1965, 1967) (Text-Fig. 2).

Because of genetic uniformity tissue and plantlets produced from the differentiation of cell clones should prove useful. However, this may no~ often be true. Melchers (1966) has shown that callus of Nicotiana tabacum

154 RAMESI-I MAHESHW,~ RI

pith and plants produ ed from it show var: ing ploidy. Such a changc is desirable in studies of the effects of ploidy on cell growth and the susceptibility of resistance of cells to a pathogen.

M I C R O C U L T U R E C H A M B E R

i ii ili !!i!i i,i ii i i ii! iil ii!ii iii i i!i !il ! i!!Fill iiiiiiii!i!!i!iiiiiiii!i!iiiii i : : ! i~? ? i:i ? i:i i!i i ??iii!}! iiiii!

i J

t !

. . . . . . . . . . . . . . ~ .. . . . . . A ......... ~ . ~ ~' . , " !~,.~i~.~;~7;"; . . . . . . . . . . , , . . . . . . . . . . . . . . . J I , I I l ; L . . . . . . . . . . . . . . l iqu id med ium with ce l ts

.~ i ~ . . . . . . . . . . . . . . . . . p a r a f f i n o i l ," t . . . . . . . . . . . . . . . . . . . . cover s l i p a 2 x z z m r n : . . . . . . . . . . . . . . . . . . . . . . cove r s l i p r i s e r a 2 x z a m m

. . . . . . . . . . . . . . . . . . . . . . . . . . . p a r a f f i n ol i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . glass sl ide a5 x 7 s m m

T~x-r-Fm. i. Micro~'Jttre chamber for growth and obs¢ixation of living, single cells, and c e l l g r o u p s . ( J o n e s etaL, Am. J. Bot., 1 9 6 0 , 9 7 , 4 6 8 - 7 5 ) .

Cell cloning has another application in plant pathology. It helps us to know whether or ro t a diseased tissue comprises entirely of cells showing abnormal physiology.

Yet another advantage is that in cultures tissues can be kept almost indefinitely alive, thus permitting many kinds of-investigations on them. White (personal communication) has grown the roots of a tomato, which is an annual plant, for over 30 years in culture as a "clone " and has defined its nutritional requirements. The famous HeLa strain of human cells, which served as substratum for much of the early work on the polio vaccine is being still used for countless expe: imental studies all over the world and has been maintained for over 20 years although the woman from whom it was isolated died of cancer for which she was operatef].

Applica:ions of Plant Tissue and Cell Culture 155

q

)

PLANTLET

ROOT FORMATION

SHOOT FORMATION

YOu)IG pLAwr s'v'tu plvH iN O-U¢o~uu U*,'ruRc PI.ANT . - -

~Ai_ItlS FROil PITH

N. TABACUU

SUSPI[NSIOI4 CULTURE

N I COT I ANA GLUTINOSA X

CALLUS F R O M ~ ¢£LL MASS ¢ CELL MASS FROM MtCROCULTUR[

@®e@ GI[LL MASS tROl l

SINOLK C I[;kl. \ Id ICR~U~,,TUR[

T~xT-FIa. 2. Plant tissue and cell culture as bsezt to demonstrate cellular totipofency. A sm',,.P, piece of parenchyma tissue ~as cut from cylinaets of pith remove t aseptically wttn the help of a sterile cork borer from a hybrid tobacco plant. This piece placed on an ae.ar meditma c. n- raining minimal salts + vitamins + coconut milk -t- 2,4-D + naphtnaleneac(tic acid (NAA), proliferated into ~ callus. Callus .,as tran>ferrvd to liquid medium and agitated on a reclploeal shaker. Callus dLsociated into c¢il grotF, and f~eo oAls in liquid medium. Single cells were picked Nith the helr~ of a micro:)i~ette and placed in a droplet of medirtm in mieroculture chamber. Di¢islc.ns in single call v, ere followed by pha~-corttr~ st oplies. Therest 'ltin~ colony con- tin ted undifferentiated growth when transferred ase)ticallt to an agar mediu;r~ containing 2,4-D, NA.A., and coconut milk. When the callus .,as transl'errea to a mediun containing IAA and kinetin in a certain ratio, many leafy sheets were differentiated. Healthy shoots sepxratod and transferrect to a basal mouium, developed roots. A plantlet was transferred to sterilized soil in pots and coeered to keep high h ;mid.,'ty during its eatl.¢ developrrent. Mat's,re plant 0erived fro.n a sin,olo cell prod.~c~d flowers and fertile seeds. (Vasil and H.ildcb[andt, Planta, 1967, 75, 139-5I.)

THE CROWN-GALL

The crown-gall disease is a classic example of the usefulness of tissue and cell culture in the study of neoplastic growth. It is caused by a tumor inducing principle (TIP) produced by the bacterium, Agrobacterium tumefaeiens. The TIP has the ability to transform normal cells to tumor

156 RAMESH MAHESHWARI

cells in short time. The tumor cells proliferate independently of the growth-restraining influences of the plant and form galls.

Proof of autonomous nature of disease.--In some plants secondary galls arise away from the site of inoculation with bacterium. By isolating tissue cultures from secondary galls they have been shown to be sterile and capable of continuous growth on a simple medium (White and Braun, 1942). When small fragments of such cultures were grafted on healthy hosts, the fragments developed into new tumors. Tissue cultures reiso- lated from these tumors and regrafted on healthy hosts also developed into tumors. This proved, according to Koch's postulates, that cells once transformed to tumor state have undergone a heritable change and can thus develop into tumors independently of the causal bacterium.

Cell transformation--a gradual and progressive process.--After its inoculation into the host the crown-gall bacterium can be selectively killed by heat treatment (Riker, I926). Thus the transformation from normal to tumor cell could be interrupted. Braun (1958) showed that the normal cells that were transformed to tumor cells 39, 50 and 72 hours after inoculation developed into tumors at slow, moderately fast and very rapid rate, respectively. Tissue cultures from each of these 3 types maintained indefi- nit, ly their characteristic growth rates. Thus the transformation of normal cells to tumor cells takes place gradually and progressively.

Metabolic markers associated with normal and tumor celts.--A clue to the differential growth rates of the three types of tumor tissue was obtained by comparing the growth requirements of the tissues (Braun, 1958). All three types grow continuously on the basal medium. The growth of the slow-growing tissues can be speeded up to the maximal rate by supplying them some special ingredients.

The normal cells, unlike the tumor cells, cannot grow on the basal medium. To achieve a growth rate similar to that of the fully transformed tumor cells, the normal cells require seven additional organic substances: myo-inositol, glutamine, aspartic acid, adenylic and cytidylic acids, an auxin, and kinetin. Thus, at least seven biosynthetic systems which are not functional in the normal cell are p~rmanently activated in the tumor cell. Except for the cell division promoting hormone, kinetin, the requirements by the normal cell for the other six supplements is eliminated by merely increasing the levels of KC1, NaNO~ and NaH~PO4 in the culture medium (Wood and Braun, 1961); i.e., increased levels of inorganic ions make the normal ceils synthesize the essential organie metabolites. This

Appliea:ions of Plant 7'issz,'e at:d Cell Culture 157

has been referred as ion-activation of some essential biosynthetic systems (Braun and Wood, 1962). The degree of activation of these systems in a tumor cell determines the rate at which the tumor cell grows. Tumor cells efficiently take up ions from dilute solutions; but normal cells do not (Braun and Wood, 1962). Also no energy requiring active transport mecha- nisms were in'volvcd in the solute uptake by tumor ceils (Wood and Braun, 1965). On the basis of these demonstrations it has been suggested that during the transfoimation process the properties of the cell membrane change and that lhe cell membrane serves as an important regulator of bios)nlhetic metabolism which determines the autonomy in the tumor cell (Braun and Wood, 1962; Wood and Braun, 1965). Plant tissue cultures have thus revealed a fundamental difference between the normal and crown-gall cell.

The nature of change.--The single-cell culture technique has provided answers to two basic questions on palhological growth. Firstly, it has pro~cd that lhe t tmor tissue is ccmposed entirely of transformed cells. This was demonstrated by raising tissue clones from cells isolated from the tumor tissue by the "nurse-culture" method (Braun, 1959). These tissue clones behaved in every respect like the parent tumor (teratoma). Secondly, the single-cell culture technique has provided a possible explanation for the growth behaviour of the transformed cells (Braun, 1959, 1965). Clones established from single cells of tobacco teratoma on a basic culture medium showed that the teratoma consists entirely of tumor cells (Text-Fig. 3, A-D). Buds formed on the teratoma clones were grafted on cut stem tip of normal tobacco plant (Text-Fig. 3, E). The graft grew into an abnormal shoot (Text-Fig. 3, F). Cells isolated from this abnormal shoot grew on lhe basic medium like the original tumor cells from which the clones were derived. Successive grafting (Text-Fig. 3, G-I) resulted in a normal shoot (Text-Fig. 3, J). This flowered and set seeds which produced normal tobacco plants (Text-Fig. 3, K, L); cells isolated from these normal plants did not grow on basic medium (Text-Fig. 3, M). Thus there was reversal of cells from tumorous to normal state.

Such a gradual recovery of tumor cells to normalcy, according to Braun (1965), rules out that the change to tumorous state is the result of mutation of genes. Rather, abnormal growth may be due to altered expres- sion of information in genes, i.e., the change is epigenetic. It must be pointed out, however, that such a reversal has been obtained only of teratoma cells which are not fully transformed tumor cells, but not of tumor cells.

158 RAMESH MAI~SIIWAI~j

The crown-gall example shows clearly how a precise control of ingredients of a tissue culture medium may pinpoint various biosynthetic differences between the healthy and the diseased cells. As chemically defined c~Iture media are available for the in vitro cultivation of many plant tissues, our understanding of cancer in plants has progressed more

ClO~e Growth on basic cutture medium

P~,r t ial cecovery of mal ignant

state

Seed ~ : a n in Plani soil

Graft cn New growth third norma! gradually f rom se~d is

plant becomes completely normal normal

M Tissue from

~ . recovered p'ant does not

---.-.-.~.~ grow on basic cultur~ medium

produced

Tsx'r-FiG. 3. Reversal of mal/gnant growth using t©ratoma of singlo.c, li origin. Slightly modified from "Tho Reversal of Ttmlor Growth," by Armin C. Braun. (Copyright 1965 by Scientific American, Inc. All rights reservod).


than that in animals. Defined nutrient media, which only permit detec- tion of metabolic shifts, are unfortunately not available for culture of most animal tissues.

VIRUS TUMORS

The tumors on Rumex induced by wound-tumor virus is another non- self-limiting disease. The fiterature on tissue culture studies in this disease has been summarized by Black (1966). Although the tumor tissue has been grown, in vitro tissue culture from healthy root has not been obtained to permit comparative studies. The interesting features of tumor tissue culture are their high phosphate requirement and extracellular secretion of s-amylase (Black, 1966).

INSECT GALLS

The most striking feature of insect galls is that their growth is deter- minate. The morphological form that a gall assumes depends upon the nature of the pathogenic insect. The galls are self-limiting structures, i.e., their growth and development are dependent upon continued stimulation by insects. In the hope that comparative tissue culture studies may reveal some fundamental differences, galls of simple structure showing little internal differentiation have been successfully cultivated in vitro. Attempts to culture highly organized cynipid galls have largely failed (Pelet, 1959). Tissue cultures derived from leaf gall of grape caused by Phylloxera and from normal grape stem tissue have been investigated in detail by Hildebrandt (1963) and his associates (Arya, 1965; Warick and Hildebrandt, 1966). Qualitative biochemical differences which may impli- cate specific compounds in gall formation or supporting abnormal growth were not found. Cellular changes may have been lost in subculturing of tissues. So far tissue cultures have not proved very useful in studies of self-limiting neoplasms.

NEMATODE DISEASES

The etiological relationship of nematodes to plants is often difficult to prove on account of contaminating bacteria and fungi. The rearing of nematodes on tissue cultures should obviate this problem. Nematodes reared in sterile conditions could form the basis of fundamental studies.

The first successful attempt to obtain an aseptic population of nematodes involved the use of roots of host seedlings grown aseptically or excised roots in culture (see Dougherty, 1960). Chen et al. (1961) reported

B5

1~0 RAMESH MAHF.SHWARI

a culture technique, using corn roots for the multiplication of Pratylen- ehus penetrans. It was possible to observe microscopically the process of infection by nematodes.

Schuster and Sullivan (1960) reported that three species of nematodes, Meloidogyne hapla, M. incognita, and Nacobbus batatiformis induced characteristic host reaction on excised tomato roots grown on agar medium. Consistent differences in the shape of epidermal cells and in the formation of root hairs at the site of development of galls were observed. This shows that secretions of these nematodes differ and they may be distin- guished by reaction of hosts grown in vitro with them.

Many types of callus tissue have proved favourable substrate for multiplication of plant parasitic nematodes. Darling et al. (1957) reported that Ditylenchus destructor, the nematode causing potato rot, grew on callus tissues of potato, carrot, clover and tobacco. The chrysanthemum nematode, Aphelenchoides ritzemabosi, is known to reproduce rapidly in callus tissues of tobacco, carrot, periwinkle and marigold (Dolliver et al., 1962). In tobacco tissue, 3,880 progenies were obtained in about a month after inoculation with one gravid female. Webster (1966) reported that callus tissues of oat, rose, apple, and potato also favoured the reproduction of this nematode. The nematodes Aphelenchoides ritzemabosi and Ditylenchus dipsaci reproduced faster in calli than in normal plant tissues (Webster and Lowe, 1966). Thus plant tissue cultures have proved ideal substrates for many plant parasi ic nematodes.

Tissue culture should prove useful in defining factors in plant tissues which influence the nematode population. For example, maximal reproduction occurred when growth of the callus tissue was best (Dolliver et al., 1962; Webster and Lowe, 1966). Barker and Darling (1965) found that the age of callus culture at the time of its inoculation with Aphelenchus avenae influenced the reproduction rate of the nematode appreciably. Repro- duction was rapid in 2-to 4-day-old cultures, slow in 7-to 10-day-old cultures and almost absent in 2-to 3-week-old cultures. They suggested that failure of nematode reproduction in old plant callus was due to the presence of thickened cell-walls in the host tissue which the nematodes could not penetrate and, therefore, could not derive nutrition.

That kinds and concentrations of inorganic ions in the host cells may be important in parasitism is suggested by the work of Dolliver et al. (1962). Tobacco cultures grown on media in which Zn, Mn, Fe, Mg or K cations were reduced by I/9 of their usual levels did not reproduce the progeny


per female Aphelenchoides ritzemabosi. But a medium low in Ca ions reduced the reproduction rate appreciably, whereas increasing the Ca 3-fold that in normal medium increased the reproduction rate. The addition of the chelating agent ethylenediaminetetraacetic acid (EDTA) to the medium markedly reduced the reproduction rate. These findings should be extended to other species of nematodes and their hosts.

Callus tissues produced in the presence of 2, 4--D are better substrates for nematode reproduction (Schroeder and Jenkins, 1963; Krusberg and Blickenstaff, 1964; Webster and Lowe, 1966). Callus tissues derived from resistant plants on media containing 2, 4-D favoured nematode reproduction. When an extract of Aphelenchoides ritzemabosi was added to the medium, it induced callus formation in red clover seedlings (Webster and Lowe, 1966). Nematodes feeding on such tissue reproduced faster than those on seedlings in vivo. Webster and Lowe suggest that substances secre- ted by nematodes into the host may act in a manner similar to 2, 4-D. The host-parasite relationship is perhaps controlled in part by the host's growth substances and in part by nematode secretion. Future researches should explain how plant tissues in the presence of plant growth regulators can have such striking effects on nematode population.

The root-knot nematode, Meloidogyne #zcognita, infected intact roots in preference to excised roots, which in turn were more favourable to nematodes than undifferentiated tissues (Sayre, t958). Using proper ratio of IAA and adenine, Sayre was able to induce vascular differentiation in tobacco callus cultures. Both differentiated and undifferentiated callus tissues were used as substrates for nematode development. Histological examination of the material showed the presence of adult female nematodes in the differentiated tissues only. Thus, the differentiation of vascular tissue appeared necessary for the complete development of the parasite. An understanding of the influence of vascular tissue in pathogenesis should prove rewarding. The experimental induction of vacularization in callus tissues by grafting shoot apices (Camus, 1949) or by the application of auxins or sugar or both (Clutter, 1960; Wetmore and Rier, 1963) should make tissue cultures ideal for such investigations.

VIRUS DISEASES

Although animal virology laas greatly progressed by application of tissue culture technique, plant virology has not benefited to the same degree by this technique. This is because plant cells unlike animal cells cannot be grown as monolayers which are important in studies of cytopathic effec,,s

162 RAMESH MAFIESHWARI

intercellular multiplication, tumorogenic properties and viral genetics. These days animal tissue cultures are being used almost exclusively in the production of vaccines against poliomyelitis, measles and adenovirus. Another obstacle in the increased use of the tissue culture technique in plant virology has been the frequent ioss of virus from infected cells in cultures (Hildebrandt and Riker, 1958; Kassanis et aL, 1958).

However, the fact that for the cultivation of plant tissues, in coa- trast to animal tLsues, chemically defined nutrient media are available has evoked interest in the studies of plant viruses through tissue culture technique. Concerted attempts to grow plant cells in monolayers are urgently needed in which our knowledge of plant cell-waU chemistry may be signi ficant.

A recent review has discussed plan viruses in tissue cultures (Ray- chaudhuri, 1966). Several workers have studied the effccts of metabolites and their ana ogs on the titer of virus and the growth of callus tissue to gain information on the syn hesis and inhibition of the infectious nucleic acid. Raychaudhuri and Mishra (1965) have demonstrated that the incorpo- ration of purine and pyrimidine bases into medium enhanced, while that of the corresponding analogs :nh!b~ted chilli mosaic virus in totacco tissue cultures.

Hirth and Lebeurier (1965) reported that tobacco tissue cultures could be infected with TMV or the ribose nucleic acid (RNA) extracted from TMV. This is a further demonstration that viral nucleic acid carries the genetic specificity to code for both self-replication and amino-acid sequence in its coat protein.

The question why callus ceils lose virus during subculturing has fund .- mental and practical importance. On the practical side it indicates the possibility of obtaining virus-free stocks of such plants as cannot be freed of virus by other methods. That not all cells in a callus tissue derived from a systemically infected plant contain the virus is now confirmed (Hansen and Hildebrandt, 1966). Single cells were mechanically separated from repeatedly transferred TMV-infected callus culture of Nicotiana tabacum. Using a microassay, involving the application of a homogenate of single cell in 0.2/zl of buffer to indicator hosts, it has been shown that in the calli derived from infected host some cells are healthy while at least 40~ carry the virus. When the calli were induced to differentiate, some healthy shoots also emerged besides the virus-infected shoots which could be

Appliea'dors o~" Plant Tissue and Cell Culture 163

subsequently infected with TMV. This proved the origin of healthy shoots to the existence of healthy single cells or cell aggregates in infected callus.

A very useful application of the suspension and callus cultures and of the microchamber technique (Text-Fig. 1) has been in the demonstration of totipotency of TMV-infected single cells (Chandra and Hildebrandt, 1967). TMV inclusion-bearing cells were individually hand-picked and cultured in microchambers. ~Ihe single isolated cells divided and formed colonies of 60-100 cells which were subsequently induced to differentiate on a medium containing IAA and kinetin. Both healthy and virus-infected plants were eventually obtained from infected single cells. This shows that infection ~ith a viral nucleic acid does not alter the morl~hogenetic potential of the cell to develop into a plant.

OBLIGATE FUNGAL PARASITES

The relat!onship of obligate fungal parasites to their hosts may be clari- fied if the parasites could be grown on host tissue cultures. Maintenance of races, study of structural relationship of host and parasite and the product on of aseptic spores and mycelia wou'd become possible In addition we can make comparative investigations on the nutritional requirements of healthy and infected tissue cultures. This may reveal the growth requirements of the obligate parasites and thus contribute to the eventual axenic culture.

Morel (1948), who pioneered inves'igations on culture of cI!igr~e fungal parasites on plant tissue cultures, found that the fresh c~,llt~s of g~pes was more susceptible to Plasmopara viticola than tissues which hzd been sub- cultured repeatedlyl The monoxenic cultures were, however, unstable and the fungus partner was finally lost. Singh (1963, 1966) cultured hyper- trophied stem and gall tissues of Ipomoea infected with Albugo ipomoeae- panduranae. The fungus developed an aerial myeelium and reproduced both sexually and asexually. The initial growth of the callus carrying the oospores was rapid as compared to the one without. Some explants l:e- came free of fungus after repeated subculture. Recently, Nakamura (1965) reported the growth of Peronospora parasitica on fresh callus produced by explants of turnip root in culture. Whether or not a continuous growth of the fungus-infected host tissue was achieved and whether the conidia could infect the callus in subcultures are not reported.

164 RAMESH MAHESHWARI

Stable monoxenic culture of the obligate-endoparasitic slime-mold, Plasmodiophora brassicae has been realized (Strandberg et aL, 1966). Infec- ted cabbage root galls were cultured on a chemically defined medium. The parasite multiplied more rapidly in tissue cultures than in intact roots and the infected host did not become free of fungus on subculturing. Stages of plasmodial growth, nuclear and nucleolar enlargement, and increased starch content were also observed in tissue cultures. No ultrastructural differences were found either in the host cytoplasm or the plasmodia in natural clubroot infectiorts and mouoxenic cultures of P. brassieae (Williams and Yukawa, 1967).

The question why infection of tissue cultures witch spores of obligate parasites has generally been uns.uccessful has been studied in rust fungi (Maheshwari et al., 1967 a, b). Working chiefly with Puecinia antirrhini and snapdragon tissue cultures, it has been demonstrated ~ a t a very low perce~atage of urediospores inoculated on tissue cultures germinated. This was attributed to an extracellular production of a substance by the host tissue culture which inhibited urediospore germination. Even when germination occurred, the germ tubes did not develop nor, really. An orderly sequence of germ tube differentiation into infection structures, viz., appres- sorium, infection peg, vesicle and infection hypha, as occurs in nature, did not occur on tissue cultures. The development of infection structures is a pre-requsiite for infection (Maheshwari, 1966). In nature their development is induced by the contact of the germ tube with the host cuticle, the constituents of surface wax stimulating germ tube differentiation (Maheshwari et al., 1967 a, b). Perhaps by manip~Aating the properties of surface of tissue cultures, infection may become feasible. There are other conside- rations also which should be borne in mind in attempting to grow obligate parasites on host tissues in vitro. As mentioned before, some studies indi- cate that a certain level of organization of cells and differentiation of organs in tis3ue cultures may be necessary for infection by nematodes (Sayre, 1958). The unpublished observations of Isaac and Smith (quoted in Allen, 1959) are also pertinent. They noted that only few colonies of Puceinia helianthi could be established on detached sun-flower cotyledons when inoculated, although the attached cotyledons became heavily infected from a similar inoculation. Detachment of the cotyledons with a part of the stem at the node, however, was sufficient to aUow heavy infection. Therefore, the success of host infection by obligate parasites may depend on internal organization of cells and tissues, metabolism of cells, and external surface of tissues (Maheshwari, 1966).

App'ications o f Plant Tis~,ue and Cell Culture 165

MICROCULTURE

Several types of microchambers have been devised to allow continuous observation of growth including cell division, maturation, and the death of single cells or groups of cells. Of these, the one devised by Jones et al. (1960) (Text-Fig. 2) is most widely used today. High magnification cinephotomicrography of cells growing in such microchambers have recorded c~rtain dyaa.uic fettures of rate of protoplasmic streaming, changes of nucleus and nucleolar volume, nuclear division, origin and development of cell plate, and several other features (Das et al., 1966). The chances of detecting such details by fixation and sectioning procedures ~ould ~,cem remote; and even if detected they might be di~smissed as artefacts. Micro- culture of both host and the parasite may permit direct examination of iafection process of living cells. Studies of host-parasite interaction at cellular level is a future possibility.

This tecknique has already proved useful in study of the kinds, formation and movements of virus inclusion l:odies in callus cells (Singh ard Hildebrandt, 1966). The influence of virulent and avirulent crown-gall bacteria oa living callus cells in microcultures has been studied (Kalil et al., 1965). A greater number of cells in culture with virulent strain i ed longer than those in culture with avirulent strain or in control cultures. Cells in culture with virulent strain contained less starch after 4 weeks than those treated with avirulent strain or in control cultures. Microculture method appear to be ideally suited for studying the influence of extracellular products of pathogens such as polysacchaIides, enzymes, a~d toxirs, on livirg cells. White (1966) has improvised a micro-perfusion chamber v, hkh pro- vides coatroUed circulation of nutrients or gases, and permits otservations under high magnification of growing cells. Two chambers can be so arran- ged as to provide flow of metabolic products from o~e to anolher. TEe effects of metabolic products of diseased cell on healthy cells can be microscopically examined for extended periods.

BIOASSAY FOR GROWTH REGULATORS

The discovery and understanding of mechanism of action of plant growth regulators, particularly the cytokinins, have been possible largely by use of plant tissue cultures. They are the most reliable bioassays for cytokinins (Letham, 1967). No other assay, chemical or biological, is as sensitive and specific. Further the tissue culture bioassays have the advantage of being conducted under aseptic conditions. The role of cytokinins

166 ~ S H M.~mSBWARI

as determinants of certain pathogen-induced abnormalities has been investigated by employing tobacco stem pith callus, tobacco pith stem segments, soybean callus or carrot root discs as bioassay systems.

The bacterium, Corynebacterium fascians, upon infection of many dicotyledonous plants induces characteristic disease symptoms such as mul- tiple buds and swollen internodes. The symptoms can be imitated by applications of kinetin (Thimann and Sachs, 1966). These investigators extracted a substance from this bacterium which on application to test plants duplicated the effect of the pathogen. A cytokinin, highly active in tobacco bioassays, was isolated from cultures of C. fascians and purified (Kl~mbt et aL, 1966). It was identified as 6-(y, ~,-dimethylallylamino) purine (Hel- geson and Leonard, 1966). It is worthwhile to mention that this cytokinin

HN--Ctt2~HC~,,C( cHs I CHs

6-('Y, ~-dirneth:/laUylamlno) purlne

HN__C~.__HC__CC cry' \CHI0tt

Z~tln

occurs next to the anticodon of serine t-RNA i~n yeast (Zach~u et aL, 1966). Another naturally occurring cytokinin, zeatin, first isolated flcm corn kernels has now been isolated from a mycorrhizal fungus, l~hizopogon roseolus (Miller, 1967).

Tissue culture bioassays have revealed increased levels of cytokinins in Plasmodiophora-induced galls on roots of Brassica rapa (Matsubara and Nakahira, 1967) and Meloidogyne incognita induced galls on tobacco root (Krupasagar and Barker, 1966). Studies on the malformations induced by insects, species of Albugo, some rust fungi, and Taphrina should prove rewarding. It may be pointed out that disturbances in levels of growth hormones, however, may not be necessarily associated with abnormal growth alone.

Excised tobacco pith tissue which has a specific requirement for both an auxin and a cytokinin for growth has been used to elucidate the mechanism of gall formation by the nematode, Meloidogyne incognita (Sandstedt and Schuster, 1966 a, b). This nematode increased growth of tissue and induced typical syncytia only when both, an auxin and a cytokinin, were present in the medium. This indicated that the nematode-induced growth


was dependent on two kinds of growth substances which were not fur- nished by the nematodes. Nematode secretions, which contain neither auxins nor cytokinins, can induce the formation of syncytia. They suggested that the nematodes feed on the syncytial contents and multiply and produce more secretions that act synergistically with auxin and cytokinin to promote further tissue growth. By growing nematodes on peeled tobacco stem segments cultured in vitro and comparing the effects induced by IAA, it was shown that abnormal growth is not due to release of bound auxins as suggested by Sayre (1958). On the contrary, the effects of root-knot nematodes in some respects resembled the effects of 2, 3, 5-triiodobenzoie acid (TIBA), an inhibitor of polar transport of auxin. It was suggested that nematodes enable the infected tissue to retain and use endogenous auxin which otherwise would be transported to other parts of the plant.

CONCLUSIONS

The usefulness of plant tissue and cell culture in plant pathology is not limited to serve as a substrate for the growth of parasites. It has, besides, given insights into the characterist cs of pathological growth, the weapons of attack of the pathogens, the factors affecting infection and multiplication of the pathogen, and how the host responds to an invading organism. Tissue and cell culture technique is not an ehd in itself. If used with other techniques, such as electron microscopy, cinephotomicrography, mutagenic radiation, radioisotopy and immunoserology, the scope of investigations would greatly enlarge. Nonetheles, the results obtained through use of tissue and cell culture must be evaluated keeping in mind that metabolism of cells grown in vitro may be different. As Penso and Balducci (1963) have s ated, " . . . Cells in culture are cells in revolt, cells that no longer obey the laws established for the organizat on of cells in tissue. " The structural simplicity of ,ssues and cells n cuLure helps to simpli y our understanding of complex phenomena occurring in the intact organism; their different physiology ra se many questions. "Tissue culture on the one hand facittitaes, extends and renovates biolog cal research and on the o her hand makes it more difficult and arduous" (Penso and Baldtacci, 1963).

ACKNOWLEDGEMENTS

I thank Drs. N. S. Rangaswamy and P. S. Ganapathy for suggesting improvements in the manuscript. Thanks are also due o the University Grants Commission for the award of a Senior Research Fellowship.

t68 RAM~H MAI~SI-IWARI

RFFERENCEg

Alien, P.J. . . "Physiology and biochemistry of defense," In Horsfall, J. G. and Dimond A. E. Editors, Plant Pathology, Acadcnlic Press, N.w York, 1959, 2, 435-67.

Arya, H. (2. . . "Cl t , . r a l behavio.r of insect gall and normal plant stem single cell don.s ," In C. V. l~nakrishnan, Editor, Tissue Culture (Prec. Se,ninar Baroda), W. Lnk , The Hag.e, 1965, 293-309.

Barker, K. R. and D~aling, H. M. "Reprod.~ction of Aphelenehus avenae on plant tit~ .es in e..iture,°~ Nematologica, 1965, I1, 162-66.

Black, L.M. . . "Physiology of vir..s ind .cod t:tmors in plan~s," In R..Mand ~ . , Editor, Handbuch der Pflcmzenphysiologie, Spfingez,

Braun, A. C.

and Lipetz J.

and Wood, H.N. . .

Carol:s, G.

Chandra, N. and I-lfldebranat, A.C.

~ : n d

Chen, Tseh-An, Kilpatrick, g. A. and Rich, A. E.

CLtteL Ma~y E. . .

Darling, H. M., Faulkner, L. R. and Wallendal, P.

Das, T. M., Hild©brandt, A. C. and Riker, A. J.

Vorlag, Berlin, 1966, 15, 246-66. . . " A physiological basis for a't~ono'no zs~growth of h =. crown-

gall t liner cell," Prec. hath. Aead Sci., U.S.A., 1958, 44, 344--49.

• . " A demonstra'ion of the recovery of the crown-gall t trnor cell with the ):so or complex tumors of single cell origin," Ibid., 1959, 45, 932-38.

. . "The reversal of tumor growth," Set. Amer., 1965, 213, 75--83.

. . "The vse of tiss e c .It .re in phttopatholoM," bt Will r, er, E. N. Editor, Cells wzd Tissues in CMtare, Acadenic

Press, London, 1966, 3, 6f2-722. "On the activation of certain ~sen'.ial biosynthetic systems

|n cells of Viaca rosea L.," Proc. natn. dcad. Sci., U.S.A., 1962, 48, 1776-82.

. . "P,.eeherehe~ s : r le role des bo:tgeom dam Its pheno.~nes de morphog6n6so," Rev. Cyt. Biol Vdg. 1949, 9, 1-191.

"Growth in raicro-~:lt:re of s;ng)e toba co cells infected with tobacco mosul: ~irt,s," Se~e~Jee, N.Y., 1956, IS:Z, 789-891.

. . "'Differentiation of ! nts frGm tobacco mosaic virt s inclusion- bearing and ind .sion-frce single tobacco cells," Virology' 1967, 31, 414-21.

"Sterile ¢~zit:re techniq:es as tools in plant ne~natology research," Ph.vtopathology, 1961, 51, 799-800.

"Hormonal ind.ction of vascdar tlssm in tobacco pith in vitro," Science, N.Y., 1960, 132, 548-49.

"Culturing the potato rot nematode (Abstr.)," Phytopathology, 1957, 47, 7.

"Cinephotomicrography of low temperat ~re effe~'s on cyto- plasmic streaming, nmlcolar activity and mitosis in single tobacco cells in microc,lt.tre," Am. I. Bat., 1966, 53, 253-59,

Applications of Plant Tissue and Cell Culture

Dolli~er, J. S., Hildebrandt, A. C~ and Riker, A. J.

Dougherty, E. C.

HabeHandt, G.

Hansen, A. J. and Hildebrandt, A.C.

Helgeson, J. P. and Leonard, N.J .

Hildebr.-.ndt, A. C.

and Piker, A. L ..

Hirth, L. and Lebeurier, G ..

3'ones, L. E., Hildebrandt, A. C., Riker, A. 3'. and Wu, J. H.

Kalil, Millicent, Hildebrandt, A. C. and Evert, R. F.

Kassanis, B., Tinsley, T. W. and Qual,, E.

Kl~nbt, D , Thies, Gail and Skc og, F.

Kr, tpa. agar, V. and Barker, K.R.

Krttsberg, L. R. and Blickenstaff, M. L.

Letham, D. S.

Libbert, E., Wichner, S., Schiewer, U.o Risch, H. and Kale, W.

169 "Stadies of reproduction of Apheleneoides ritzemabost,

Schwartz on plant tiss-~es in c.!tarc," Nematologica, 1962, 7, 294-300.

. . "C_ltivation of Aschelminths es2ecially rhabditid nematodes," In Sasser, J. N. and Jenkins, W. R., Editors, Nematology, Fandamentals and Recent Advawes with Emphasis on Pla~u Parasitic a d Soil Forms, The University of North Carolina Press, Chapel Hill, 1960, 297-318.

. . "Kaltarberasche mit isolierten P~anzenzeilen," Sitzur.gsber. Akad. der Vdtss. Wien, Math. Natarwiss., 1902, 111, 69-29.

"The distribatlon of tobacco mosaic virJs in plant callas calt~res," Virology, 1966, 28, 15-21.

"Cytokinins: identification of compo:nds isolated from Cory,.ebacterium fascians,'" Prec. natn. Acad. Sci., U.S.A., 1966, 56, 60-63.

.. "Growth of single cell clones of diseased and normal tiss~e origins," In Plant Tissue CMture and Morphogenesls, Scholar's Library, New York, pp. 1963, 1-22

"Vira~ses and single cell clones in plant tissue cl lture," Fed. Prec., 1958, 17, 986-93.

"Remarques sur la sensibilit6 des celLdes des c=:ltt:res de tissas de tabac ~, l'infection par le viras de la mosaique da tobac on son acide riboaacleiq:e," Rev. g~n. Bct., 1965, 62, 5-20.

"Growth of somatic tobacco cells in microeulture," Am. d. Bet., 1960, 47, 468-75.

"The influence of virulent crown-gall bacteria and non-virulent Agrobacterium radiobacter on starch metabolism in tomato cells in mic.rocultvre (Abstr.)," Ibid., 1965, 52, 633.

"The inoc :lation of tobacco callus tissue with tobacco mosaic viras," Ann. appl. 1Siol., 1958, 46, II-19.

"Isolation of cytokinins from Cor2nebacterium fas¢ians,'" Prec. ham. Acad. Sci., U.S.A., 1966, 56, 52-59.

"Increased cytokinin concentrations in tol~acco infected with root-knot nematode Meloidogyne incognita (Abstr.)," d~hytopatholog~, 1966, 56, 885.

"Inflnence of plant growth regulating s'abstances on repro- daction of Ditylenchus dipsaci, Pmtylenchus penetrans and Pratylenchus zeae on alfalfa tissae ceiteres," Nemato- logiea, 1964, 10, 145-50.

. . "Chemistry and physiclogy of klnetin-like compounds," A. Rev. PI. Physiol., 1967, 18, 349-64.

"The influ-nce of epiphytic bacteria on auxin metaboliam~" Pfi, nta, J~966, 68~ 327-34.

170 L~bert, E., Wichner, S.,

Kaiser, W., Manicki, A., • Man,eaffel, R., Riecko, E.

and SchrOder, 1L Maheshwari, 1L

~ , Allen, P. ~I. and Hildebrandt, A. C.

~ , Hildebrandt, A. C. and Allen, P. J.

Mats:~bara, S. and Nakahira, R.

Melchers, G.

Miller, C. O.

Mountain, W. B.

Mot, l, G. . .

M~ir, W. H., Hildebrandt, A. C. and R.iker, A. J.

Nakamara, H. . .

Pelet, F.

Penso, G. and Balducci, D.

Raychaadhari, S. P.

- - - - and Mishra, M. D.

Riker, A. J.

R A M ~ H MAHESHWARI

"Aaxin content and anabolisra of sterile and non-sterile plants, w/th special regard to the infl:,ence of epiphyti¢ bacteria (Abstr.l," Sixth International Conference on Plant Growth Substances, Carleton University, ~967.

. . "The phjsielogy of penetration and infection by .,redic- s~ores of r ,st f mgi," Ph.D. Thesis, University of Wisconsin, Madison, 1966.

"Physical and che~aical factors controlling the development cf infection str~.ct~res from urediospore germ t.,bes of rust f~ngi," Phytopathology, 1967 a, 57, 855-62.

"Factors affecting the growth of "r.st fmgi on host tiss'ae c..lt.,res," Bet. Gaz., 1967b, 128, 153-59.

"C:'tokinin activity in an extract from the gall of Plasmodio. ph~ra-infected root of Brassiea rapa L.'" Bet. Mug. Tokyo, 1967, 80, 373-74.

. . "Gonetioal aspects in call .s c ltare work fAbstr.)," Inter- national Symposi'am on Plant Pathology, New Delhi, Bull. laban Phytopath. See., 1968 (In press).

. . "Zeatin and zeatin riboside from a mycorrhizal fungus," Science, N.Y., 1967, 157, 1055~57.

. . "Acceptable standards of proof and approaches for evaluat- ing plant-nematode relationships," In Sa~ser, J. N. and Senkins, W. K., Editors, Nematology, F~;damentals and Recem Advances with Emphasis on Plant Parasitic and Soil Forms, The University of North Carolina Press, Chapel Hill, 1960, pp. 422-25.

"Kecherches s'~r la c .lt.*re associee do Parasites obHgatoires ot do tissus vegetaux," Ant.. EMphMies, 1948, 14, 123-234.

"The preparation, isolation and growth in ¢~alture of single cells from higher plants," Am. J. Bet., 1958, 45, 589--97.

"'The "ase of tiss.e cult::res in the study of obligate parasites," In White, P. g . and Glove, A. IL, Editors, Prec. later- national Conference on Plant Tissue Culture, McOltchan P,tbEshing Corp., Berkeley, 1965.

. . "Growth in vitro of grape, elm, poplar, willow and oak tissues isolated from norm~ 1 stems and insect galls," Ph.D. Thesis, University of Wisconsin, Madison, 1959.

. . Tissue Cultures in Biological Research, Elsevier Publishing Co., Amsterdam, 1963.

. . "Plant ,/irases in tissue Galtare," Adv. Virus Res., 1966, 12, 175-206.

. . "Effect of some metabolites and their analogues on the infectivity of tooacoo tissae culture containing chilli mosaic viras," Virology, 1965, 25, 483-84.

. . "Sradies on the inflaence of some environmental factors on the development of orown-gall," J. agr/c, Res., 1926~ $7, ~5-$L

Applications of Plant Tissue and Cell Culture Sandstedt, R. and Schuster,

M.L.

17i "Excised tobacco pith bioassays for root-knot nematode-

prod, ted plant growth substances," Physiol. Plant., 1966a, 19, 99-104.

and ~ - - . . "The role of a'Lxins in root-knot nomatcde4nduced growth on ©xciscd tobacco stem segments," Ibid., 1966b, 19, 960--67.

Sayre, R.. M. .. "Plant tis%~e culture as a tool in the study of the physiology of root-knot nematode Meloidogyne incognito Chit," Ph.D. Thesis, University of Nebraska, Lincoln, 1958.

Schroeder, P. H. and Jenkins, "R.eprodtction of Pratylenchus penetrans on root tissues. W.R.. grown on three media," Nematologica, 1963, 9, 327-31.

Sch2st~r, M. L. and S.11ivan, T. "Species differentiation of nematodes thro:~gh host reaction in tissue cult.re. I. Comparisons of Meloidogyne hapla, M. incognita and Nacobbus batatiformis," Phyto- pathology, 1960, 50, 874--75.

Singh, H. .. "The growth of Alb~go in the call "-s ~alture of lpomoea," Curt. ScL, 1963, 32, 472-73.

. . "On the variability cf the call~-s of lpomoea-infeeted with Alb,~go grown ~.mder ir vitro conditions," Phytomorphology, 1966, 16, 189-92.

Singh, M. and Hilde~randt, A. C. "Movemems of tobacco mosaic vir.:s incLsion bodies within tobacco c',.llus cells," Virology, 1966, 30, 134-42.

Steward, F. C., Kent, Ann E., "The cult .re of free plant cells and its significance for embryo- and Mopes, Marion O. logy and morphogenesis," In Monroy, A. and Moscona,

A. A., Editors, Current Topics in Developmental Biology, Academic Press, New York, 1966, pp. 113-54.

Strandberg, J. O., Williams, P.H. "Monoxenic cuk ~re of Plasmodiophora brassicae with cabbage and Y.kawa, Y. tiss.e (Abstr.)," Phytopathclogy, 1966, $6, 903.

Thimann, K. V. and Sachs, T. "The role of eytokinins in the ' fa :dat ion ' disease caused

, Groehowska, M. and Avadhani, P. N.

Vasil, Vimla and Hildebrandt, A.C.

and

Warick, R.. P. and Hildebrandt, A.C.

Webster, J .M. ..

.... and Lowe, D.

by Corynebacterium fascians," Am. J. Bet., 196% 53, 731-39.

"The role of tryptophan as an I.A.A. pree~.zrsor (Abstr.),'" Sixth lt.ternational Conference on Plant Growth Substances, Cerleton University, 1967.

"Differentiation of tobacco plants flora single, isolated cells in microcaltare," Science, N.Y., 1965, 1.50, 889-92.

"Further st-:dies on the growth and differentiation of single isolated cells of tobacco in vitro," Planta, 1967, 75, 139-51.

"Free amino-acid contents of stem and Phylloxera gall tissue cultures of grape," PI. PhysioL, 1966, 41, 573-78.

"Production of oat callus and its susceptibility to a plant parasitic nematode," Nature, Lena., 1966, 212, 1472.

"The effect of the synthetic plant-growth substance, 2,4- diehlorophenoxyacetic acid, on the host-parasite relationship of some plant parasitic nematode in monaxenic culture," Parasitology, 1966, $6, 312-22.

t"I2 ~ a MxI-mmwgtu

Wetmoro, R. H. and Bier, J. P,

White, P. K.

and Braun, A. C..

Williams, P. H. and Yukawa, Y.B.

Wood, H. lq. and Braun, A. C.

a n d ~

Zaehau, H. G., Dutting, D. and Feldman, H.

"Experimental Induction of vascular tissues in callus of angiosperms," Am..1. Bet., 1933, 50, 418-30.

. . "Versatile ~erfusion chamber for living cells and organs," Science, N.Y., 1966, 152, 1758-60.

" A cancerous neoplasm of plants: Autonomous bacteria- flee crown-gall tissue," Cancer Res., 1942, 2, 597-617.

"Ultra.truetural studies on the host-parasite relations of Plasmodiophom brassieae," Phytopathology, 1967, 57, 682-87.

"Studies on the regulation of certain essential biosynthetic systems in normal and crown-gall tumor cell," Prec. natn. Acad. Sci., U.S.A., 1961, 47, 1907-13.

. . "Studies on the net uptake of solutes by normal and crown- gall t~mor cells," Prec. hath. Acad. Sci., U.S.A., 1965, 54, 1532-38.

"Nu¢leotide sequence of two serine-specific transfer tibonueleic avid," Angew. Chem. lnternat. Edit., 1966, 5. d22.

EXPLANATION OF PLATE

PLATE IV

FIe. 1. Technfque dovdoped by Muir etaL ('1958) for culturing isolated single cells. A. A ~,inglo coil is placed on the uppel s~rface of a filter-paper square whoso lower smface makes good contact with an activo!y growing "nurse" tissue of the same straiv. B-C. The cell di~;d~-s and forms a colony of coils. D. A clone dedvoa from a single coil cominues ;ndercmlen" g~owlh ~hon transferred to nut~.;ent agar medium. (Hildobraadt and Biker, Federation Plot., 1958, 17~ 986-93 0

Ramesh Maheshwari Proc. Indian Acad. Sci,, B, Vol. LXIX, Pl. IV

Fro. 1

Applications of plant tissue and cell culture in the study of physiology of parasitism

Documents

Transcript of Applications of plant tissue and cell culture in the study of physiology of parasitism