Planta (Ber].) 73, 28--36 (1967)

SHORTENING THE JUVENILE PHASE FOR FLOWERING I N K A L A N C H O E P I N N A T A PERS. MRIDUL WADHI and H. Y. MOHAN RAM Department of Botany, University of Delhi, Delhi 7, India Received August 2, 1966 Summary. Plants of Kalanchoe pinnata flower normally at the end of 2 years. Flowering in the juvenile phase (3- and 9-month-old plants) has been induced by application of gibberellin (GA) either to the shoot tip and the youngest pair of leaves, or to the third leaf. Three-month-old plants required more exogenous GA (50 ~g/ plant) than 9-month-old plants (5 ~g/plant). The simultaneous application of the growth retardant (2-chloroethyl)trimethylammonium chloride = CCC via the roots did not interfere with GA-induced flowering but overcame the inhibitory effects produced by a high concentration of GA (150 vg/plant) when applied alone. Introduction Flowering plants pass through a series of distinct phases during their development. T h a t phase during which a shift from the vegetative to the reproductive state cannot be induced (even under conditions otherwise favourable for flower formation) is the juvenile phase. I t is also called minimum vegetative stage, ripeness-to-flower stage, immature condition, or sterile stage. I n some plants the juvenile phase can be shortened either by increasing the light intensity (HIGAZY, 1962), or by apphcation of auxin (DE Z•EUW and LEOPOLD, 1955). There is evidence t h a t applied gibberellins m a y also shorten the juvenile phase (KATo et al., 1958; Rv, SV,~DE and VIA~A, 1959; PENNER, 1960; PHA]~IS et al., 1965). Under the conditions of Delhi, plants of Kalanchoe pinnata Pets. (syn. BryophylIum calycinum Salisb.) flower at the end of the second year, after having borne at least 37 pairs of leaves. The objective of this investigation was to reduce the juvenile phase in this plant b y gibberellin treatment and to correlate the flowering response with the amount of gibberelhn used. An a t t e m p t was also made to study Yfgibberellin-indueed flowering in juvenile plants was in any way affected by the growth retardant, (2-ehloroethyl)trimethylammonium chloride (CCC). Material and Methods Mature leaves of K. pinnata 1 were planted in pots and were maintained under field conditions of light and temperature prevailing from February, 1964 to March, 1965 in the Botanical Garden, University of Delhi. By proper manipulation of the i This plant is easily propagated by leaf cuttings.

Shortening the Juvenile Phase in Kalanchoepinnata

29

propagation time, plants in the juvenile phase (3 and 9 months old) were obtained for treatment in the first week of November, 1964. The 3-month-old plants were 3.6~0.5 cm tall with 6 pairs of leaves; the 9-month-old plants were 36.7• cm tall with an average of 12 leaf pairs. In one set of experiments aqueous solutions of gibberellin Aa (gibberellic acid; GA)2, containing Tween 80 (0.05 %) as wetting agent, were applied to the shoot tip and the youngest leaf pair of 3- and 9-month-old plants. Four concentrations of GA (1, 3, 10 and 30 y,g/0.1 m]) were used and 0.1 ml applied per treatment. Five treatments were given, on alternate days, the total amount of GA received by each plant thus being 5, 15, 50 or 150 ~g respectively. Controls were treated with distilled water containing Tween 80. In another set, GA was applied to 9-month-old plants through the third leaf (from the apex). The leaf was first washed with a dilute solution of a detergent, Teepol B-300, and rinsed with distilled water. An aqueous solution of GA (15, 20, 25 and 30 rag/l) was applied to either surface and the leaf was enclosed within a polythene bag into which 10 ml of GA of the corresponding concentration were poured. The bag was tied around the petiole by a thread, with the lamina dipping into the liquid. Leaves of control plants were washed with Teepol B-300 and were enclosed in bags containing distilled water. In the first and second sets of experiments, 10 plants were used per treatment. In the third set of experiments 9-month-old plants were given simultaneously 100 ml of CCCa at a concentration of 2000 rag/l, applied through the soil, and 0.1 ml of GA solution (1, 3, 10 and 30 ~g/0.1 ml), applied to the shoot tip and the youngest pair of leaves enclosing it. Five treatments were given on alternate days and 5 plants were used per treatment. Thus, each plant received 1000 mg of CCC and 5, 15, 50 or 150 ~g of GA. At the beginning of the experiment the youngest visible leaf pair was marked as leaf pair No. 0. At the end of the experiment the number of leaf pairs and bracts developing above the marked pair were counted. The number of flowers produced was also recorded.

Results U n d e r t h e c o n d i t i o n s e x i s t i n g in Delhi, p l a n t s of Kalanchoe pinnata s t a r t f l o w e r i n g b y t h e e n d of N o v e m b e r w h e n t h e d a y l e n g t h a v e r a g e s l l h o u r s ~. D u r i n g t h i s p e r i o d t h e a v e r a g e d a y t e m p e r a t u r e is n e a r l y 26~ a n d n i g h t t e m p e r a t u r e goes d o w n to 9~ Flowering continues through t h e m i d d l e of M a r c h . F l o w e r d e v e l o p m e n t is n o r m M u p to a n t h e s i s a n d

pollination but ovaries and ovules remain arrested, not developing into fruits and seeds.

Application o/GA to Shoot Tix and Youngest Lea/ Pair. T r e a t m e n t w i t h G A p r o d u c e d t h e u s u a l v e g e t a t i v e responses, t h a t is, l i g h t e r leaf colour, i n c r e a s e in h e i g h t a n d i n t e r n o d e l e n g t h , a n d also a n i n c r e a s e in l e a f - p M r number. 2 GA was obtained from the ImpericM Chemical Industries Ltd., Welwyn, England. 3 This substance was kindly supplied by American Cyanamid Co., Stamford, Conn., USA. a We have not done any experiments to establish its precise photoperiodic requirement. I t may be either a short-day or a long-short-day plant.

30

lV[.WAD~r[and H. Y. iVIo~A~RAM: Shortening the Juvenile Phase

Three-month-old controls and plants treated with 5 ~zg and 15 ~zg of GA per plant did not enter the reproductive phase but some of the plants treated with 50 ~zg and 150 ~zgformed flowers after 6 or 7 weeks (Fig. 1 A). More flowers were produced on plants treated with 150 ~zg than with 50 f~g of GA (Table 1). The flowers were morphologically similar to those normally found in 2-year-old plants. Table 1 Influence o/GA* on stem growth and/lowering in 3-month-old Kalanchoe pinnata plants Height at treatment: 3.6-4-0.5 cm. Number of leaf pairs at treatment: 6. Growth period: Three months after treatment. Total GA per plant (~zg)

Total height (cm)

0"** 5 15 50 150

14.8-t-0.8 27.0=t=2.0 28.4=t=2.2 29.8• 38.5=k 3.6

No. of leaf pairs** after treatment

5.2=[=0.3 6.2=k 0.6 7.3-4-0.9 9.9-b 0.9 11.2~ 0.9

No. of plants floweringJ-

No. of flowers per plant

0 0 0 5 6

0 0 0 25 33

*Applied five times to shoot tip and youngest pair of leaves. ** Including leaves and bracts. *** Received distilled water containing Tween 80. t Out of ten plants. I n 9-month-old plants, as little as 5 ~zg/plant of GA were sufficient to induce flowering 4 weeks after treatment (Fig. 1 B). The number of plants flowering at this concentration was 7 but at the other concentrations all 10 plants came to flower. The controls remained vegetative throughout (Table 2). The number of flowers produced per plant increased with an increase in the GA dosage (Table 2), the greatest number developing at 50 ~zg/ plant (Fig. 2A). At 150 ~zg/plant, the flower buds showed symptoms of misdevelopment (Fig. 2 B); probably in connection with this, the number of flowers was lower than that with 50 ~zg of GA. Otherwise, the development of the flowers was comparable to that in 2-year-old plants. Foliar Application o[ GA. The flowering response of 9-month-old plants in which GA was supplied through the leaf was comparable to that in 9-month-old plants treated with GA at the shoot tip. The optimum Fig. 1A--C. Induction of flowering in 3-month-old (A) and 9-month-old (B) plants of Kalanchoe pinnata by GA application to shoot tip and youngest pair of leaves, and in 9-month-old plants by foliar application of GA (C). Photographed 2 months after treatment. A, • 0.1; B, • 0.07 ; C, • 0.08

Fig. 1 A - - C (legends see p. 30)

32

M. WADm and H. Y. MOHANRAil:

concentration of GA, as assayed by stem elongation, number of flowering plants and number of flowers per plant (Fig. 1C), was 15--20 rag/1. At 25 rag/1 and 30 rag/l, both stem length and flower number tended to be lesser.

E//ect o / C C C and GA on Stem Growth and Flowering. Nine-month-old plants treated with CCC alone remained in the vegetative state and stem

Fig. 2A and B. Portions of inflorescences developed on 9-month-old plants after application of 50 ~g (h) and 150 ~g (B) of GA to shoot tip and youngest pair of leaves. The development of flower buds is arrested in B. Photographed 2 months after treatment. • 0.5

length and'leaf number were not affected (Table 3). Application of GA to CCCrtreated plants resulted in marked internodal elongation, increase in leaf number, appearance of the characteristic light green colouration of the foliage, and initiation of flowering. Partial flowering occurred with as little as 5 fxg of GA per CCC-treated plant. I n the previous experiments, where GA alone had been applied to the shoot tip of 9-month-old plants, it was observed t h a t 150 [• of GA proved toxic to the development of flower buds. I n combination with CCC, flowering was normal and there was an increase in the number of flowers (Tables 2 and 3).

Shortening the Juvenile Phase in Kalanchoe pinnata

33

Table 2

In/luence o/ GA * on stem growth and/lowering in 9-month.old Kalanchoe pinnata plants Height at treatment: 36.7• Number of leaf pairs at treatment: 12. Growth period: Three months after treatment. Total GA per plant (~g)

Total height (cm)

No. of leaf pairs ** after treatment

No. of plants floweringr

No. of flowers per plant

0"** 5 15 50 150

39.5=[_2.0 50.3-~ 5.0 82.0=[=2.2 76.1• 64.4•

4.8• 6.4~: 0.9 7.1=k 0.1 7.3• 7.0•

0 7 10 10 l0

0 30 51 69 59

* Applied five times to shoot tip and youngest pair of leaves. ** Including leaves and bracts. *** l~eceived distilled water containing Tween 80. ? Out of 10 plants. Table 3. Flowering response and stem growth o/ 9-month-old Kalanehoe pinnata

plants treated with CCC and GA Height at treatment: 36.7• Number of leaf pairs at treatment: 12. Growth period: Four months after treatment. Total amount of chemicals applied/plant CCC * (g)

0 1 1 1 1 1

Total height (cm)

No. of leaf pairs** after treatment

No. of No. of plants flowers floweringt per plant

44.6~ 5.6 46.2~: 2.5 70.0 ~ 8.7 80.6~= 2.5 80.4=~ 3.3 95.6 ~ 7.9

0.6~: 0.5 5.8~: 0.4 7.8 ~ 0.2 8.6~ 0.2 8.2~ 0.4 8.6 ~= 0.2

0 0 3 5 5 5

GA (ttg)

0"** 0"** 5 15 50 150

0 0 26.8 57.0 76.0 104.0

* Supplied through roots in aqueous solution. ** Including leaves and bracts. *** Received distilled water containing Tween 80. t Out of 5 plants. Discussion T h e j u v e n i l e phase of perennials m a y e x t e n d from a few m o n t h s to several years; in annuals a n d biennials its d u r a t i o n is m a x i m a l l y a few m o n t h s . I n some annuals like Pharbiti# nil (KtrJIRAI an d I~[AMU~A, 1958) t h e p h o t o p e r i o d i c stimulus m a y be p er cei v ed e v e n b y t h e cotyledons. I n Chenopodium rubrum (CuMMING, 1959) t h e m i n i m u m n u m b e r of leaves necessary for t h e plants to flower is two. I n B r y o p h y l l u m daigremontianum t h e j u v e n i l e phase m a y last for one year (PlaNNIng, 1960) or 3

Planta (Berl.), Bd. 73

34

M. WADm and H. Y. MO~L~N1%~:

two years (see ZE]~VAA~T, 1962) and the plants must bear at least 10--12 pairs of leaves before they are ready to flower (ZEEvA)aCT, 1962). The plants of Kalanchoe pinnata flower in nature, under the conditions of Delhi, only when they are 2 years old and have produced at ]east 37 pairs of leaves. ZEEVAART (1958) suggested the following alternative explanations for the existence of a juvenile phase: (i) The leaves on the juvenile plant are less sensitive to photoinduction and therefore either fail to produce the floral stimulus, or are producing it in insufficient quantities; (2) the terminal and axillary buds are unable to respond to the floral stimulus. Evidence in support of the first proposal comes from work on Perilla crispa (ZEEvAART, 1958) and Lolium temulentum (EvAns, 1960). ZVmVAA~T (1962) showed that the second supposition was untenable in Bryophyllum daigremontianum. By grafting juvenile plants onto flowering donor stocks he demonstrated that the growing tips were fully capable of responding to the floral stimulus. We have found that juvenile plants of K. pinnata flower in response to GA treatment. Our experiments do not permit, however, a decision as to whether this flowering response is due to increased flower-stimulus production by the leaves or to increased sensitivity of the buds. The demonstration of naturally occurring gibberellin-like substances in the plants (see KNAvP, 1963), induction of flowering by plant extracts containing gibberellins (see LAI~G, 1965), and increase in the endogenous levels of gibberellins during the change from the vegetative to the reproductive phase (ItAgADA and NITSCH, 1959; LA~G, 1960; ~TICttOLLSand LA~G, 1964; S~E~E and LA~G, 1964) suggest that there is a definite relationship between flowering and gibberellin metabolism. The failure of flower induction in the juvenile phase could be attributed either to a total absence or to an insufficient quantitiy of native gibberellins. In agreement with these ideas is the report of I~]~SENnE and VIA~A (1959) who obtained induction of flowering by GA application in seedlings of Bryophyllum proli/erum which normally require 6 years to flower. Three-month-old plants of B. daigremontianum required as little as 0.15 ~g of GA to enter the reproductive phase (PE~NEI~, 1960). KATO et al. (1958) induced cone development (flowering) in seedlings and adult plants of Cryptomeria japonica by GA treatment. PHAI~IS et al. (1965) obtained similar results in Cupressus arizonica and found, moreover, that younger seedlings required more exogenous GA than older ones. The present results with K. pinnata also show clearly that younger plants (3-month-old) need more exogenous GA than older ones (9-month-old), namely, 50 ~zg as compared to 5 tzg. These findings, therefore, suggest that younger plants have low endogenous levels of GA which are insuffi-

Shortening the Juvenile Phase in Kalanchoe pinnata

35

cient for flower formation. T h u s i t is possible to induce flowering in t h e juvenile p h a s e b y exogenous s u p p l y of GA. T h e g r o w t h r e t a r d a n t CCC induces effects opposite to those of GA. I n r e c e n t y e a r s KENDE et aI. (1963) a n d NINNEMA~N et al. (1964), w o r k i n g w i t h cultures of F u s a r i u m monili]orme h a v e d e m o n s t r a t e d t h a t CCC does n o t d e s t r o y or i n a c t i v a t e p r e f o r m e d G A b u t blocks its biosynthesis. HARADA a n d LANG (1965) a n d BALDEV et al. (1965) came to t h e conclusion t h a t t h e s a m e m e c h a n i s m is o p e r a t i v e in higher p l a n t s a n d accounts for t h e effects of CCC a n d certain o t h e r r e t a r d a n t s on g r o w t h a n d o t h e r d e v e l o p m e n t a l processes in these plants. Our results also indicate t h a t CCC does n o t interfere w i t h exogenously a p p l i e d GA. T h e y are in a g r e e m e n t w i t h t h e conclusion t h a t juvenile p l a n t s of K. pinnata are deficient in endogenous G A since in such p l a n t s a p p l i c a t i o n of an i n h i b i t o r of G A biosynthesis should h a v e little if a n y effect on g r o w t h a n d development.

Acknowledgements. We are thankful to the late Professor P. MAttES~WARI, F.t~.S., for his keen interest in this research, to Dr. P. S. GA~-APAT~Yfor his comments, and to the Council of Scientific and Industrial Research, New Delhi and the University Grants Commission for the award of a research fellowship to one of us (M. W.).

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M. WAD~ and H. Y. 1V[OttANRAM: Shortening the Juvenile Phase

LA~G, A.: Gibberellin-like substances in photoinduced and vegetative Hyoscyamus plants. Planta (Berl.) 54, 498--504 (1960). - - Physiology of flower initiation. In: Encyclopedia of plant physiology (W. Ru~LAND, ed.), vol. XV/1, p. 1380--1536. Berlin-Heidelberg-New York: Springer 1965. ~N~ICKOLLS,P. B., and A. LANG: Endogenous gibberellin changes and internode growth in the barley inflorescence. Plant Physiol. 89 (Suppl.), XXVI (1964). NIC~EMA~r H., J. A. D. ZEEVAA~T,H. KE~DE, and A. LANG: The plant growth retardant CCC as inhibitor of gibberellin biosynthesis in Fusarium monili]orme. Planta (Berl.) 61, 229~235 (1964). P E ~ R , J.: Uber den Einflul~ yon Gibberellin auf die photoperiodisch bedingten Blfihvorg~nge bei Bryophyllum. Planta (Bet1.) 55, 542--574 (1960). P~ARm, R. P., M. D. E. RSDDAT, C. C. PHILLIPS, and E. HEFTMA~: Precocious flowering of Arizona cypress with gibberellin. Canad. J. Bot. 48, 923--927 (1965). RESE~CDE,F., and M. J. Vs Gibberellin and sex expression. Port. Acta biol. A 6, 77--98 (1959). SKE~CE,K. G. M., and A. LAN@:Native gibberellinsand flower formation in Bryophyllure daigremontianum. Plant Physiol. 89 (Suppl.), XXXVII (1964). ZEEUW, D. DE, and A. C. LEOPOLD: Altering juvenility with auxin. Science 122, 925--926 0955). ZEEVAART,J. A. D.: Flower formation as studied by grafting. Meded. LandbHoogeschool Wageningen 58 (3), 1--88 (1958). - - The juvenile phase in Bryophyllum daigremontianum. Planta (Berl.) 58, 543--548 (1962). Dr. M~IDULWADHI and Dr. H. Y. MOItANI~AM Department of Botany, University of Delhi Delhi-7, India