Planta (Berl.) 81,372--375 (1968)
Short Communication
Induced Morphogenesis in Tissue Cultures of Solanum xanthocarpum P. S. RAo a n d S. Na~AYA~ASWAMI Biology Division, Bhabha Atomic Research Centre, Bombay, India l~eceived February 24~, 1968
Summary. Differentiation of shoots and roots or unorganized proliferation could be induced in tissue cultures of Solanum xanthocarpum when callus tissues habituated to a medium containing 2.4-D and meso-inositolwere transferred to an auxinfree medium or to a medium in which the ratio of auxin and meso-inositol had been altered. As a p a r t of a n overall p r o g r a m m e of research concerned with phenom e n a of growth a n d differentiation i n p l a n t tissues c u l t u r e d i n vitro, tissue cultures of a n u m b e r of higher p l a n t s of economic i m p o r t a n c e a n d of medicinal value such as Ocimum, Fagopyrum, Cicer, Tylophora, Datura a n d others were established i n this laboratory. The present report deals with a n example of the control of organogenesis i n tissue cultures of Solanum xanthocarpum SCttt~AD a n d W~NDL, b r o u g h t a b o u t b y sequential changes in the n u t r i e n t conditions a n d exogenous auxin. Defoliated shoots, excised from plants of S. xanthocarpum growing wild and from aseptically germinated seedlings, were cultured on White's medium (WRITE, 1943) containing coconut water (10%, v/v) and 2.4-dichlorophenoxyacetic acid (2.4-D; 6 rag/l) to obtain a dedifferentiated tissue. The cultures were kept under continuous light, at 25 • 2~ and a relative humidity of 50 to 60%. Proliferation of the explants occurred within 3 weeks of incubation, resulting in the formation of a mass of callus (Fig. 1 a). Tissue cultures thus initiated were maintained successfully through repeated subcultures of the adult callus at intervals of one month on the medium of ~UtCASttIGE and SKOO~ (1962) fortified with coconut water (10%, v/v), 2.4-D (1 rag/I), adenine sulphate (5 rag/l), meso-inositol (100 rag/l) and acid-hydrolysed casein hydrolysate (200 rag/l). The tissue was yellowish-white and parenchymatous, but not very friable. During a period of 4 weeks, the cultures have yielded a 6- to 8-fold increase in fresh weight. W i t h a view to i n d u c i n g organ differentiation a n d to ascertaining the role of growth substances i n v o l v e d i n this p h e n o m e n o n , tissue e x p l a n t s of u n i f o r m size isolated from 3-week-old stock callus were grown on the basal m e d i u m to which growth regulators like 1-naphthalcneaeetic acid (NAA), gibberellie acid (GAs), k i n e t i n a n d 3-indoleaeetic acid (IAA) were added, either singly or i n c o m b i n a t i o n . I n some experiments the c o n c e n t r a t i o n s of a d e n i n e sulphate, 2.4-D a n d inositol were varied.
Induced Morphogenesis in Tissue Cultures of Solanum xanthocarpum
373
Fig. l a - - d . Morphogenesis in tissue cultures of Solanum xanthocarpum, r root; sh shoot, a Undifferentiated mass of callus, x 1.2. b Organization of roots. X 1.5. e Organization of roots and shoots, x 1.2. d Development of shoots without roots. • A d d i t i o n of G A a (2 nag/l) p r o v e d i n h i b i t o r y for t h e g r o w t h of callus whereas N A A (1 mg/1) a n d casein h y d r o l y s a t e (800 nag/l) p r o m o t e d cell proliferation. Conabinations of kinetin a n d I A A , or adenine s u l p h a t e
374
P. S. RAo and S. ~NTARAYANASWA~II:
with IAA in varying concentrations caused no differentiation but only enhanced unorganized growth. However, on transfer of tissue to basal medium in which meso-inositol was increased to 400 rag/1 and the concentration of 2.4-D was lowered to 0.1 or 0.2 mg/1, organ formation was observed. Numerous roots differentiated within a week after the transfer, distributed irregularly over the culture (Fig. 1 b). This was followed by the organization of a number of shoot primordia which later developed into stems and leaves (Fig. 1 c). If, however, the tissue was cultured on basal medium from which 2.4-D had been excluded but in which mesoinositol (400 rag/l) was retained, shoots were formed but no roots (Fig. 1 d). Root formation was also observed in cultures of the buckwheat, F a g o p y r u m esculentum, when the tissues which had been growing on a basal medium containing auxin (2.4-D) and inositol were transferred to an auxin-free medium (SAYAGAV~EI~and N~mAYA~ASWA~, unpublished data). Induction of organ formation in tissue cultures is a complex process brought about by the interaction of a number of factors. Some callus tissues tend to lose their ability to initiate organs on repeated subculture (Mo~EL, 1948; JACQUIOT, 1955; MrLLW~and SKooo, 1953; M~YE~, 1956; ToR~wy, 1959). GAUTHE~T (1966) explains this phenomenon "as being due to the loss of a specific factor contained in the primitive explant which is no longer synthesized in vitro". The experimental findings reported here are of special interest in that incorporation of a balanced ratio of growth supplements or the deletion of one or the other in the basal medium could bring about cellular differentiation leading to organ formation in callus tissues which were under continued subculture and had apparently lost their morphogenetie potentialities. I n the present instance withdrawal of 2.4-D from the medium to which the tissue had been habituated could trigger the processes leading to the formation of shoots, or both shoots and roots, or to unorganized proliferation. Studies along these lines should prove useful in arriving at meaningful generalisations about morphogenesis in plant tissue cultures.
References
GAUT]~:ERET,R. J. : Factors affecting differentiation in plant tissues grown in vitro. In: Cell differentiation and morphogenesis, p. 55--95. Amsterdam: NorthHolland Publ. Co. 1966. JACQUIOT,C. : Formation d'organes par le tissu cambial d' Ulmus campestris, L. et de Betula verrucosa, Gaerth. cultiv~s in vitro. C.R. Acad. Sci. (Paris) 240, 557--558 (1955). MAYnR,L. : Wachstum und Organbildung an in vitro kultivierten Segmenten yon Cyclamen persicum. Planta (Berl.) 47, 401--446 (1956).
Induced Morphogenesis in Tissue Cultures of Solanum xanthocarpum
375
MILLER, C. 0., and F. SKOOG: Chemical control of bud formation in tobacco stem segments. Amer. J. Bot. 40, 768--773 (1953). MOREL, G. : Researches sur la culture associ6e de parasites obligatoires et de tissus v6g6taux. Ann. ]~piphyt., N. S. 14, 123--234 (1948). MVl~ASIZlOE,T., and F. SKOO~: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum (Cph.) 15, 4 7 3 4 9 7 (1962) TOm~EY, J. G. : Experimental modification of development in the root. In: Cell organism and milieu (D. RvD~ICI~, ed.), p. 189--222. New York: Ronald 1959 WItlTE, P. R. : A handbook of plant tissue culture. Lancaster, Pa. : Jacques Catte 1943. Dr. P. S. RAo and Dr. S. NAI~AYA~ASWAMI Biology Division Bhabha Atomic Research Centre Trombay, Bombay-74, India
Short Communication
Induced Morphogenesis in Tissue Cultures of Solanum xanthocarpum P. S. RAo a n d S. Na~AYA~ASWAMI Biology Division, Bhabha Atomic Research Centre, Bombay, India l~eceived February 24~, 1968
Summary. Differentiation of shoots and roots or unorganized proliferation could be induced in tissue cultures of Solanum xanthocarpum when callus tissues habituated to a medium containing 2.4-D and meso-inositolwere transferred to an auxinfree medium or to a medium in which the ratio of auxin and meso-inositol had been altered. As a p a r t of a n overall p r o g r a m m e of research concerned with phenom e n a of growth a n d differentiation i n p l a n t tissues c u l t u r e d i n vitro, tissue cultures of a n u m b e r of higher p l a n t s of economic i m p o r t a n c e a n d of medicinal value such as Ocimum, Fagopyrum, Cicer, Tylophora, Datura a n d others were established i n this laboratory. The present report deals with a n example of the control of organogenesis i n tissue cultures of Solanum xanthocarpum SCttt~AD a n d W~NDL, b r o u g h t a b o u t b y sequential changes in the n u t r i e n t conditions a n d exogenous auxin. Defoliated shoots, excised from plants of S. xanthocarpum growing wild and from aseptically germinated seedlings, were cultured on White's medium (WRITE, 1943) containing coconut water (10%, v/v) and 2.4-dichlorophenoxyacetic acid (2.4-D; 6 rag/l) to obtain a dedifferentiated tissue. The cultures were kept under continuous light, at 25 • 2~ and a relative humidity of 50 to 60%. Proliferation of the explants occurred within 3 weeks of incubation, resulting in the formation of a mass of callus (Fig. 1 a). Tissue cultures thus initiated were maintained successfully through repeated subcultures of the adult callus at intervals of one month on the medium of ~UtCASttIGE and SKOO~ (1962) fortified with coconut water (10%, v/v), 2.4-D (1 rag/I), adenine sulphate (5 rag/l), meso-inositol (100 rag/l) and acid-hydrolysed casein hydrolysate (200 rag/l). The tissue was yellowish-white and parenchymatous, but not very friable. During a period of 4 weeks, the cultures have yielded a 6- to 8-fold increase in fresh weight. W i t h a view to i n d u c i n g organ differentiation a n d to ascertaining the role of growth substances i n v o l v e d i n this p h e n o m e n o n , tissue e x p l a n t s of u n i f o r m size isolated from 3-week-old stock callus were grown on the basal m e d i u m to which growth regulators like 1-naphthalcneaeetic acid (NAA), gibberellie acid (GAs), k i n e t i n a n d 3-indoleaeetic acid (IAA) were added, either singly or i n c o m b i n a t i o n . I n some experiments the c o n c e n t r a t i o n s of a d e n i n e sulphate, 2.4-D a n d inositol were varied.
Induced Morphogenesis in Tissue Cultures of Solanum xanthocarpum
373
Fig. l a - - d . Morphogenesis in tissue cultures of Solanum xanthocarpum, r root; sh shoot, a Undifferentiated mass of callus, x 1.2. b Organization of roots. X 1.5. e Organization of roots and shoots, x 1.2. d Development of shoots without roots. • A d d i t i o n of G A a (2 nag/l) p r o v e d i n h i b i t o r y for t h e g r o w t h of callus whereas N A A (1 mg/1) a n d casein h y d r o l y s a t e (800 nag/l) p r o m o t e d cell proliferation. Conabinations of kinetin a n d I A A , or adenine s u l p h a t e
374
P. S. RAo and S. ~NTARAYANASWA~II:
with IAA in varying concentrations caused no differentiation but only enhanced unorganized growth. However, on transfer of tissue to basal medium in which meso-inositol was increased to 400 rag/1 and the concentration of 2.4-D was lowered to 0.1 or 0.2 mg/1, organ formation was observed. Numerous roots differentiated within a week after the transfer, distributed irregularly over the culture (Fig. 1 b). This was followed by the organization of a number of shoot primordia which later developed into stems and leaves (Fig. 1 c). If, however, the tissue was cultured on basal medium from which 2.4-D had been excluded but in which mesoinositol (400 rag/l) was retained, shoots were formed but no roots (Fig. 1 d). Root formation was also observed in cultures of the buckwheat, F a g o p y r u m esculentum, when the tissues which had been growing on a basal medium containing auxin (2.4-D) and inositol were transferred to an auxin-free medium (SAYAGAV~EI~and N~mAYA~ASWA~, unpublished data). Induction of organ formation in tissue cultures is a complex process brought about by the interaction of a number of factors. Some callus tissues tend to lose their ability to initiate organs on repeated subculture (Mo~EL, 1948; JACQUIOT, 1955; MrLLW~and SKooo, 1953; M~YE~, 1956; ToR~wy, 1959). GAUTHE~T (1966) explains this phenomenon "as being due to the loss of a specific factor contained in the primitive explant which is no longer synthesized in vitro". The experimental findings reported here are of special interest in that incorporation of a balanced ratio of growth supplements or the deletion of one or the other in the basal medium could bring about cellular differentiation leading to organ formation in callus tissues which were under continued subculture and had apparently lost their morphogenetie potentialities. I n the present instance withdrawal of 2.4-D from the medium to which the tissue had been habituated could trigger the processes leading to the formation of shoots, or both shoots and roots, or to unorganized proliferation. Studies along these lines should prove useful in arriving at meaningful generalisations about morphogenesis in plant tissue cultures.
References
GAUT]~:ERET,R. J. : Factors affecting differentiation in plant tissues grown in vitro. In: Cell differentiation and morphogenesis, p. 55--95. Amsterdam: NorthHolland Publ. Co. 1966. JACQUIOT,C. : Formation d'organes par le tissu cambial d' Ulmus campestris, L. et de Betula verrucosa, Gaerth. cultiv~s in vitro. C.R. Acad. Sci. (Paris) 240, 557--558 (1955). MAYnR,L. : Wachstum und Organbildung an in vitro kultivierten Segmenten yon Cyclamen persicum. Planta (Berl.) 47, 401--446 (1956).
Induced Morphogenesis in Tissue Cultures of Solanum xanthocarpum
375
MILLER, C. 0., and F. SKOOG: Chemical control of bud formation in tobacco stem segments. Amer. J. Bot. 40, 768--773 (1953). MOREL, G. : Researches sur la culture associ6e de parasites obligatoires et de tissus v6g6taux. Ann. ]~piphyt., N. S. 14, 123--234 (1948). MVl~ASIZlOE,T., and F. SKOO~: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum (Cph.) 15, 4 7 3 4 9 7 (1962) TOm~EY, J. G. : Experimental modification of development in the root. In: Cell organism and milieu (D. RvD~ICI~, ed.), p. 189--222. New York: Ronald 1959 WItlTE, P. R. : A handbook of plant tissue culture. Lancaster, Pa. : Jacques Catte 1943. Dr. P. S. RAo and Dr. S. NAI~AYA~ASWAMI Biology Division Bhabha Atomic Research Centre Trombay, Bombay-74, India
