Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 99, No.3, May 1990, pp. 257-265. © Printed in India.

Insect induced plant galls in tissue culture U KANT and VIDYA RAMANI Plant Pathology and Tissue Culture Laboratory, Department of Botany, University of Rajasthan, Jaipur 302004, India Abstract. Plant tissue culture enabling the assessment of excised organ tissues and cells and the effect of various metabolites and gall and normal tissues are discussed with reference to insect induced plant galls. In particular the stem and rachis galls of Prosopis cineraria caused by a chalcid and Lobopteromyia prosopidis, stem gall of Emblica officinalis Gaertn induced by a Lepidopteran, Betousa stylophora Swinhoe, stem gall of Zizyphus mauritiana induced by a mite, Eriophyes cernuus, the Phylloxera gall on grape and many others are discussed. Keywords. Plant galls; tissue culture; Prosopis cineraria; Lobopteromyia prosopidis; Emblica officinalis; Betousa stylophora; Zizyphus mauritiana; Eriophyes cernuus; Phylloxera.

1. Introduction

Galls ,are manifestations of growth, positive 'or negative and of abnormal differentiation induced on a plant by animal or plant parasites (Meyer 1987). They may arise on shoots, roots, petioles, stipules, leaf-blades, vegetative and flower buds, inflorescence axis, bracts, flowers, fruits, etc. Agencies causing galls include physical and chemical agents, genetic constitution, bacteria, viruses, fungi, nematodes, insects and mites. Although excellent reviews have been contributed in the field of gall induction (Miles 1968), ecology (Mani 1964, 1973) and gall histopathology (Rohfritsch and Shorthouse 1982), the factors involved in gall development need satisfactory answers. Certain critical questions involved in tumor induction with reference to nature of incitant, tissues affected, the stimulus that keeps them growing and the mechanism by which the abnormal growth can be stopped, remain to be answered. According to Norris (1979) insects probably utilise their own specific and general chemicals in the induction of a gall. Tissue culture technique has been used in a variety of ways in physiological, cytological, biochemical and morphogenetic problems of plants. The in vitro culture of tissues provides a system in which many variables can be controlled, enabling the assessment of excised organs, tissues and cells. Plant tissue culture offers certain advantages over the intact plant as an experimental material (Hildebrandt and Riker 1947; Bouckaert and Vendrig 1981). The basic idea.of tissue culture was conceived in the beginning of the 20th century. Haberlandt (1902), a German botanist has been credited with putting the technique into action. The continuous growth of isolated root tips for unlimited periods was successful first with the work of White (1939). Subsequently, successful callus cultures were established from carrot by Gautheret (1939) and Nobecourt (1939) and from tobacco by White (1939) independently. Among the first tissues grown in culture was the genetic tumor tissue of Nicotiana hybrid (Nicotina glauca x N. langdorffii) (White 1939). Extensive studies 257

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have been made with this tissue culture, including work on tumorigenesis and fine structure (Skoog 1944; Chilton et al 1977). Gall tissue of unknown origin on white spruce was isolated and studied (Reinert and White 1956). Tissue of normal and disease origin may now be grown for several years by regular transfer to fresh medium. 2. Tumor tissues in culture

The use of plant tissues to study tumorigenesis complements investigations of transformed animal cells. Moreover, because plant cells are usually totipotent, the important phenomenon of reversal of tumorigenesis may be investigated most readily in plants. In addition to the bearing on the cancer problem, plant tumor experimentation promises to yield information on the control mechanisms that restrain and regulate growth of normal plant tissues (Gordon 1981). -- Gautheret (1955) has noted that when normal cells are grown in culture some cells spontaneously grow without exogenously supplied hormones. Such 'habituated tissues' may be isolated from a variety of plant species. Since the phytohormone content of these tissues is of the same order of magnitude as in some crown gall tissues, it seems that the galls have the capacity to produce these materials (Kulescha 1952). Thus, these cells express one of the traits characteristic of crown gall tumor tissue. It is also possible to obtain cultures that are auxin or cytokinin habituated. Treatment with animal carcinogens cause tobacco tissues to lose their requirement for cytokinin (Bednar and Linsmair-Bednar 1971). Thus it is possible to switch on and switch off the biosynthetic pathways leading to the synthesis of phytohormones in non-tumorous tissues. Reviews covering the state of plant tumor field before the recent flurry of plasmid DNA oriented work can be found in Schilperoort (1969). An important review in the field before the 1970s is found in a monograph edited by Braun (1972). More recent developments have been reviewed by Lippincott and Lippincott (1980), Schell and Van Montagu (1978) and David and Tempe (1987). A few studies have also been made with viral tumor tissue cultures. Black (1949) isolated tissues from root tumors of Rumex acetosa. Detailed nutritional and other studies with virus tumor tissue have been done (Nickell and Burkholder 1950). Suspension culture was established many years after the establishment of static culture (Muir et al 1954). Single cells were grown and single cell clones were established (Muir et al 1958). Growth and tissue formation from single geranium cells and from virus infected geraniums have been studied (Kant and Hildebrandt 1971). Isolation, culture and fusion of protoplasts have lately been possible (Cocking 1960; Street 1973). The composition of the medium is critical for growth. Earlier work with tissue culture provided valuable information regarding media employed and types of tissues and organs cultured. The work has been reviewed in detail (Gautheret 1955; Street 1973). 3. Culture of imect gall tissues A number of insect-induced gall tissues have been isolated and successfully cultured in vitro. Many insect-induced plant gall tissues especially from woody plants were

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successfully cultured (Hildebrandt et al 1956; Arya et al 1962a,b; Arya 1965a, b; Kant and Arya 1969; Kant and Singh 1976; Arora and Kant 1979; Kant 1985). It has been shown that insect-induced plant gall tissues grew better on media supplemented with coconut-milk or coconut-milk with 2,4-dichlorophenoxy acetic acid (2, 4-D) and ct naphthalene acetic acid (NAA) (Hildebrandt et al 1956). Nutritional and other requirements of Phylloxera gall and normal grape stem tissue have been examined in detail (Pelet et al 1960; Arya 1965a, b). Excellent work with single cell clones of Phylloxera gall and normal grape stem tissue have been done (Arya 1965a, b). Studies related to nutrition and growth have been made for normal and gall tissues of Zizyphus jujuba Lamk. (Kant and Arya 1969; Vyas 1971; Tandon et al 1976). Tandon et al (1976) have studied some aspects of gall induction on Zizyphus. Kant

if» Figures 1-3. 1. Rachis gall of P. cineraria induced by L. prosopidis. 2. Stem gall of E. officinalis induced by B. stylophora. 3. Stem gall of Z. mauritiana induced by E. cernuus.

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and Singh (1976) have studied the effects of vitamins, choline chloride on gall and normal tissues of Z. jujuba. Studies related to nutrition and growth have been made for gall and normal tissues of Emblica o.fJicinalis (Arora and Kant 1979). Kant (1967) studied the morbid anatomy of some insect induced galls, common to Rajasthan. The cultures of Z. jujuba gall and normal tissue were established on a semisynthetic medium (Kant and Arya 1969). Subsequently, cultures of Zizyphus gall and normal tissue were established on a synthetic medium (Murashige and Skoog 1962) by Vyas (1971). Basic physicochemical, carbohydrate, nitrogen, vitamin and growth factor requirement of normal and gall tissues in culture were determined. Arora and Kant (1979) have studied the physiology of E. officinalis normal and gall tissues in culture. Singh (1978) observed the radiation effects on Zizyphus gall and normal tissues and cells. Ranwa (1983) has carried out further studies on the physiology and biochemistry of gall and normal tissues of E. o.fJicinalis. Chatterjee

Figures 4-4). 4. Normal and gall callus of E. officinalis. (A) Normal callus, (8) Gall callus. 5. IAA-kinetin interaction of gall callus tissue of P. cineraria. From left to right increasing concentrations of IAA in mgjI. From bottom to top increasing concentrations of kinetin in mg/l, 6. A single cell of gall callus of P. cineraria. ( x 400).

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(1984) has studied stem gall of Prosopis cineraria in culture. Kant and Ramani (1988a) have carried out in vitro studies on rachis gall of P. cineraria. Most of the work on galls have been carried out in economically important plants and some semi-arid zone trees. Galls studied in vitro include the stem and rachis gall of P. cineraria (a tree of the semi-arid zone) induced by a chalcid and the insect Lobopteromyia proscpidis , Mani respectively; the stem gall of E. officinalis induced by Betousa stylophora Swinhoe, the stem gall of Zizyphus mauritiana Lamk. induced by Eriophyes cernuus, a mite. The following important aspects of these galls have been dealt with: (i) Isolation of normal and gall tissues and their establishment on a suitable medium. (ii) Carbohydrate and nitrogen metabolism of gall and normal tissues. (iii) Auxin, cytokinin and phenol metabolism of tissues. (iv) Vitamin metabolism. (v) Cytomorphological studies of gall and normal tissues. (vi) Biochemical studies in vitro (and in vivo). 3.1

Isolation and establishment of normal and gall callus

Isolation of gall callus was carried out in the following manner. Young galls were longitudinally split into two and the insect was removed. Split gall pieces were treated with mercuric chloride solution (0'1%) for 15-20 min. Sterilized gall pieces were maintained and subcultured on suitable media (generally MS. medium) supplemented with growth regulators. Similarly, the normal callus was isolated from the normal counterpart, and maintained on MS medium.

3.2 Nutritional studies Nutritional studies revealed differential behaviour of normal and gall tissues to various sources of carbohydrates, nitrogen, vitamins, auxin, cytokinin and phenolics. Very little growth of tissue was recorded in the absence of these metabolites. Auxinkinetin interaction has also been studied (Kant and Ramani 1987). Partial cytokinin autotrophy has been recorded for the rachis gall tissues of P. cineraria. The effect of various phenolic substances viz. t-einnamic acid, ferulic acid, p-hydroxybenzoic acid, caffeic acid, catechol etc. on tissue growth bas been studied. None of the phenolics were essential for tissue growth, although .very low concentrations 1-1'0 mg/l) of cinnamic acid sustained good growth of gall tissue. p-hydroxybenzoic acid and catechol inhibited growth of tissues.

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3.3 Cytomorphological studies Cytomorphology of normal and gall callus cells was carried out by the microculture chamber method of Jones et al (1960). The microculture chamber method has made it possible to isolate and examine, living cells of higher plants, in an aseptic slide culture. Under sterile conditions. two drops of mineral oil were spaced on either side of the slide and sterilized cover slips placed on each drop to provide two risers to

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support a third cover slip over the cell culture. A drop of the liquid medium suspension of cells (gall and normal on separate slides) was placed between the two risers, a mineral oil dam added around the cell suspension, and a third sterile cover slip laid on the two risers and over the cell suspension. Single cells may also be isolated and grown in the microchamber. By this method, cytomorphology of normal and gall callus cell was studied under a phase contrast microscope. It revealed differences in the relative percentages, shapes and sizes of cells at different phases of tissue growth, between normal and gall tissues. Hypertrophied nuclei and cellular hyperplasy were evident in the gall callus cells of these plants (Chatterjee 1984). 3.4 I solation of single cell clones Clones of tissues may be established from single cells isolated from callus cultures of higher plants using a filter paper-nurse culture method (Muir et al 1954). A small piece of sterile filter paper was first placed on an established piece of nurse tissue growing in a flask for two or three days until it had become moistened by the nutrients from the host tissue piece. The piece of filter paper was then placed in a sterile petri plate on agar medium besides a spread suspension of cells or a callus piece. A single cell was then selected under the binocular microscope and transferred to the filter pa~r and then the filter paper with the cell returned to the nurse-piece in the culture flask. The single cell or the resulting cell mass may be examined for growth at any interval by removing the filter paper with its cell or cells to agar in a petriplate under a microscope. It may be necessary to transfer the filter paper with the small group of cells to several fresh nurse-pieces as the nursepiece ages or becomes dry. Eventually, a mass of cells of sufficient size develops that will grow when transferred directly to the agar medium. This constitutes a clone of tissue of single cell origin. This method has opened new areas for study of tissues of normal and diseased (or galled) origin at the cellular level. Single cell clones of many plants have been established. Phylloxe,.-a (gall) single cell clones have been established as mentioned earlier. 3.5

Bichemical studies

Biochemical studies revealed in general, that the gall -tissue contained more carbohydrates and reducing sugars. Gall tissues of Z. mauritiana, E. officinalis and stem gall tissue of P. cineraria have revealed hyperauxiny (Kant and Ramani 1988a,b) with the exception of rachis gall of P. cineraria (Ramani et al 1989). Changes in the activity of various enzymes viz.amylase, indole-3yl acetic acid (fAA) oxidase, phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase (TAL), peroxidase, and polyphenol oxidase have also been recorded. While the gall tissues showed a marked decrease in IAA oxidase, and polyphenol oxidase activities, with varied activity of peroxidase (Kant and Ramani 1988a, b), high amount of total phenolics and O-dihydroxy phenols were recorded in the gall tissues. Unregulated synthesis of auxin protectors (O-dihydroxy phenol) have been held responsible for hyperauxiny. These substances prevent fAA destruction by inducing a lag period in the oxidation of fAA, thus causing hyperauxiny and abnormal proliferation of the gall tissue.

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Role of oxidative enzymes and phenolics in hyperauxiny and abnormal growth in many other insect and mite induced galls such as Achyranthes aspera (Shekhawat et al 1978), Cordia myxa (Ramawat et al 1979), Camellia sinensis and Elaeocarpus lancifolius (Joshi et al 1985)have been established. 4. Conclusion

Most of the galls studied have shown increased production of growth hormones, particularly auxin and cytokinin in diseased cells. The tumefacient properties reported for Zizyphus gall tissue incited by a plant mite (E. cernuus) has given new dimensions to the problem of gall formation in plants. Crucial research needs to be directed towards understanding the molecular mechanism of insect and mite induced gall formation in plants. References Arora D K and Kant U 1979 Effect of auxins on the growth of Emblica ojficinalis Gaertn. stem gall and normal tissues in vitro; in Recent researches in plant sciences (ed.) S S Bir (New Delhi, Ludhiana: Kalyani Publishers) pp 395-399 Arya H C 1965a Cultural behaviour of insect gall and normal plant stem single cell clones; in Tissue culture (ed.) C V Ramakrishnan (The Hague; Dr W Junk Publishers) pp 293-304 Arya H C 1965b Vitamins as growth factors for Phylloxera gall and grape stem single cell clones in tissue culture; Indian J. Exp. Bioi. 3 126-129 Arya H C, Hildebrandt A C and Riker A J 1962a Clonal variation of grape stem and Phylloxera gall callus growing in vitro in different concentrations of sugar; Am. J. Bot. 49 369--372 Arya H C, Hildebrandt A C and Riker A J 1962b Nucleic acids in callus tissues from grape stem and Phylloxera gall; Phyton 19 27-29 Bednar T Wand Linsmair-Bednar EM 1971 Induction of cytokinin independent tobacco tissues by substituted fluorescences; Proc. Natl. Acad. Sci. USA 58 1178-1179 Black L M 1949 Virus tumours; Surv. Bioi. Proq. 1 155-231 Bouckaert U A M and Vendrig J C 1981 The influence of plant growth regulators on crown-gall initiation on cotyledonary leaves of Helianthus giganteus L. in vitro; Z. Pflanzenphysiol. 10375-81 Braun A C 1972 The relevance of plant tumour systems to an understanding of the basic cellular mechanisms underlying tumorigenesis; Prog. Exp. Tumour Res. 15 165-187 Chatterjee G 1984 In vivo and in vitro growth, physiology and morphogenesis of Prosopis cineraria Linn. Druce gall tissues, Ph. D. Thesis. University of Jodhpur, Jodhpur Chilton M D, Drummon M H, Merlo D J, Sciaky D, Montoya A L, Gordon M P and Nester E W 1977 Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown-gall tumorigenesis; Cell 11 263-271 Cocking E C 1960 A method for the isolation of plant protoplasts and vacuoles; Nature (London) 187 927-929 David C and Tempe J 1987 Segregation of T-DNA copies in the progeny of a regenerated plant from a mannopine positive hairy root-line; Plant Mol. Bioi. 9 585-592 Gautheret R J 1939 Sur la possibilite de realiser la culture indefinie des tissu de tubercules de carotte; C. R. Acad. Sci. (Paris) 208 118-121 Gautheret R J 1955 The nutrition of plant tissue cultures; Annu. Rev. Plant Physiol. 6433-484 Gordon M P 1981 Tumor formation in plants; Biochem. Plants 6 531-570 Haberlandt G 1902 Kultinversuche mit isolierten; Pjlanzellen. Sber. Akad. Wiss. Wien.• 111 69-92 Hildebrandt A C and Riker A J 1947 Influence of some growth regulating substances on sunflower and tobacco tissue, in vitro; Am. J. Bot. 34 421-427 Hildebrandt A C, Riker A J and Klemmer H W 1956 Growth in vitro of insect gall tissue and normal tissue on coconut milk or kinetin media (Abstr.); Plant Physiol. (Suppl.) 31 38 Joshi S C, Tandon P and Rajee A L S 1985 Changes in certain oxidative enzymes and phenolics 'in Camellia sinensis and Elaeocarpus Iancifolius leaf roll-galls; Cecid. Int. 6 51-57

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Jones L E, Hildebrandt A C, Riker A J and Wu J W 1960 Growth of somatic tobacco cells in microculture; Am. J. Bot. 47 46s--475 Kant U 1967 Studies on the insect and mite induced plant galls, Ph. D. thesis, University of Rajasthan, Jaipur Kant U 1985 Effect of phenols on growth, sugar and phenolic contents of insect induced gall and normal tissues of Emblica officinalis in tissue culture; Cecid. Int. 6 27-30 Kant U and Arya H C 1969 Growth of Zizyphus jujuba Lamk. gall and normal stem tissues in tissue culture; Indian J. Exp. Bioi. 7 37-39 Kant U and Hildebrandt A C 1971 Growth and tissue formation from single Geranium cells and from virus infected Geraniums; In vitro 7 17-20 Kant U and Singh S 1976 Effects of choline chloride and inositol on gall and normal tissues of Zizyphus mauritiana in tissue culture; A"nu. Meet. Indian Phytopathol. 1975-76, Delhi, p. I Kant U and Ramani Vidya 1987 Auxin-kinetin interaction on the growth of tumor tissues of Prosopis cineraria in tissue culture; in Proc. Natl. Symp. Tissue Cult. Econ. Imp. Plants (ed.) G M Reddy (Secunderabad: Vecon Printers) pp 331-335 Kant U and Ramani Vidya 1988a Insect-induced galls of certain economically important plants of arid and semi-arid regions; in Dynamics of insect-plant interaction (eds) T N Ananthakrishnan and A Raman (New Delhi: Oxford and IBH) pp 165-176 Kant U and Ramani Vidya 1988b Gall disease of certain economically important tree species of Rajasthan; J. Tree Sci. 7 41-44 . Kulescha Z 1952 Reserches sur l'elboration des substances de croissance par les tissues Vegetaux; Rev. Gen. Bot. 59 19 Lippincott J A and Lippincott B B 1980 Comparative pathology of abnormal growth (New York: Raven Press) Mani M S 1964 Ecology of plant galls. (The Hague: Dr W Junk Publishers) Mani M S 1973 Plant galls of India (New Delhi: MacMillan) Meyer J 1987 Plant galls and gall inducers (Berlin, Stuttgart: Gebruder Borntrager) Miles P W 1968 Insect secretion in plants; Annu. Rev. Phytopathol. 6 137-164 Muir W H, Hildebrandt A C and Riker A J 1954 Plant tissue cultures produced from single isolated cells; Science 119 877-878 Muir W H, Hildebrandt A C and Riker A J 1958 The preparation, isolation and growth in culture of single cells in higher plants; Am. J. Bot. 4S 589-597 Murashige T and Skoog F 1962 A revised medium for rapid growth and bio-assays with tobacco tissue cultures; Physiol. Plant. 15473--497 Nickell L G and Burkholder P R 1950 A typical growth of plants. II. Growth in vitro of virus tumours of Rumex in relation to temperature, pH and various sources of nitrogen, carbon and sulphur; Am. J. Bot. 37 538-547 Nobecourt P 1939 Sur la perennite et l'augmentation de volume des cultures de tissue, Vegetaux; C. R.. Seances Soc. Bioi. Paris iJO 1270-1271 Norris D M 1979 How insects induce disease; in Plant disease, an advanced treatise IV (cds). J G Horsfall and E B Cowling (New YQrk: Academic Press) pp 239--255 Pelet F, Hildebrandt A C, Riker A J and Skoog F 1960 Growth in titro of tissues isolated from normal sterns and insect galls; Am.i. Bot. 47 186-195 Ramani Vidya, Kant U and Qureshi M A 1989 Auxin profile of gall and normal tissues of Prosopis cineraria (Linn.) Druce induced by Loboperomyia prosopidis Mani in vitro and in vivo; Proc. Indian Acad. Sci. (Plant Sci). 99 385-389 Ramawat KG, Purohit S D and Arya H C 1979 Altered state of oxidising enzymes and phenolics in Cordia gall; Trans.Tsdt. Ucds.4 3s--41 . Ranwa N S 1983 Studies on galls of some economically important plants induced by insects.; Ph. D. thesis, University of Rajasthan, Jaipur Reinert J and White P R 1956 The cultivation in vitro of tumor tissues and normal tissues of Picea glauca; Physiol. Plant. 9 117-189 Rohfritsch 0 and Shorthouse J D 1982 Insect Galls; in Molecular biology of plant tumours G Kahl and J S Schell (New York: Academic Press) pp 131-152 SChell J and Van Montagu M 1978 Plant tumor research: An eoaluation; Embo Workshop, Noordwij Kerhout, Netherlands (Suppl.). Schilperoort R A 1969 Investigation on plant tumors, Crown gall. On the biochemistry of tumor induction of Agrobacterium tumefaciens; Ph. D. thesis, University of Leiden, The Netherlands

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Shekhawat N S, Ramawat K G and Arya H C 1978 Carbohydrate, protein, phenols and enzymes (PPO, PRO and IAA oxidase) in gall and normal tissues of Achyranthes aspera Linn.; Curro Sci. 47 780-781 Singh Saroj 1978 In vitro studies on normal and mite induced gall tissues of Zizyphus, Ph. D. thesis, University of Rajasthan, Jaipur Skoog F 1944 Growth and organ formation in tobacco tissue cultures; Am. J. Bot. 31 19-24 Street H E 1973 Plant ceIl cultures, their potential for metabolic studies; in Biosynthesis and its control in plants (ed.) B V Milborrow (London: Academic Press) pp 93-125 Tandon P, Vyas G Sand Arya H C 1976 Mechanism of in vitro gall induction in Zizyphus jujuba Lamk.; Experientia 32 563-564 Vyas G S 1971 On the biology of Zizyphus gall and normal stem tissues in culture, Ph. D. thesis, University of Rajasthan, Jaipur White P R 1939 Potentially unlimited growth of excised plant callus in an artificial nutrient; Am. J. Bot. 2659-64