InJIan
J.
Plant PhyskJl. Vol.xXxIi
No.4, pp 299-305 (becember, 1989)r
,Cht
ENDOGENOUS LEVEL OF HORMONES IN DEVELOPING GRAPE SEED (VITlS VINIFERA LINN)
S.N. PANDEY AND RANnT SINGH
Division of Fruits and Horticultural Technology, I.A.R.I., New Dclhi·ll0012
Received on 22 April, 1989
SUMMARY
The developing seeds of grape (cvs. Black Muscat and Iulski Muscat) showed a high level of endogenous auxins, gibberellins and cytokinins like substances during the 3rd and 4th week after anthesis, which decreased thereafter during the 5th and 6th week. Again an increase in the levels of auxins and gibbCrellins was noticed during the 7th and 8th week. The cytokinins disappeared during the 5th week and were not traced at any stage up to the ripening of berries. The level of inhibitors increased progressively from 3rd week onwards and reached highest level as the berry ripened. Except for the 7th week when the neutral auxins predominated at almost all Rf zones, the acidic auxins
were dominant at all stages and at almost all Rf zones in both the cultivars.
INTRODUCflON
Dormancy in mature grape seed has generally been related to accumulation of A8A-like inhibitors and water soluble phenolic substances (Angelova and Lilov. 1980., Kachru et al., ]969). On the other hand, freshly extracted seeds from ripe berries lack gibberellic acid (Chohan and Dhillon, 1976). Obviously, the hormonal balance required for germination of seed is already disturbed at this stage and grape seeds germinate only when dormancy is broken with stratification. which could also be substituted partially by gibberellic acid (Randhawa and Negi, 1964). Gibberel lic acid (1500 ppm) when combined with kinetin (2500 ppm) substituted stratification completely and gave over 90% germination (Pandey, 1982). It was considered necessary to know the status of growth promoting and inhibiting substances in deve loping seeds to understand the problems of seed dormancy and germination more clearly.
MATERIAL AND METHODS
100
S.N. PANDEY AND IlANJIT SINGH(butanol soluble. water soluble)-like substances and inhibitors in the developing seeds. The extracted seeds were washed and kept on moistened filter paper in a petri dish. Three lots of seeds, five grams each, were taken. One lot was crushed in 80% methanol for the bioassay of auxins and inhibitors, the second lot was crushed in 80% ethanol for cytokinins and the third in 80% acetone for gibberellins. The extraction and other operations during the bioassays were performed under green light (546 nm) in a dark room at 25±2°C temperatures. The chromatograms were run in isopropanol ammonia
+
water (10:1:1 v/v) by adopting the descend ing technique. Auxins were bioassayed using Avena sativa L. 'Kent' coleoptile straight growth test. GibberelIins were bioassayed with the modified technique of Ogawa (1963) using paddy variety Tainon-3. Cytokinins were bioassayed using radi~h (Raphanus sativus L.) cotyledon growth test as described by Letham (1968) and inhibitors by cress (Lepidium sativum L.) seed germination test.RESULTS AND DISCUSSION
The levels of endogenous auxins. gibberellins, cytokinins and inhibitors in developing seed at different stages are shown in Fig. 1 to 4.
Auxin-like substances
Higher levels of auxins (both acidic and neutral) at most of the Rf zones was observed in the beginning (3rd and 4th week) in both cultivars but reduced to minimal at the end. The level of auxins d,::creased during the fifth and sixth weeks and the acidic auxins appeared to dominate at almost all Rf zones during this period. The level of auxins decreased to 14% in Black Muscat and 20 in lulski Muscat during the 5th week and to 14
%
in both cultivars during the 6th week, aU at Rf 0.4. During the 6th week, some Rf zones were observed to be inhibitory to the neutral auxins.ACIOIC
BLACK MUSCAT
NEUTRAL
R.O-I TO 1'0 J:OR ALL SHlGES Fig. 1. Endogenous level of
• 7 8 \0 11 auxins in seeds at
{WEEKS At:TeR ANTHESIS )
various stages.
4 5 : , JUlSKI MUSCAT
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ItORMONES.IN DllVELOPING GRAPB SEE!)
Fig. 2. Endogenous level of
w~~m~~wr
gibberellins in seedsRf 0-'\ TO 1-0 FOR· ...LL STAGES
'0 at various stages
5 . a
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4
WEEKS AFTEA' ANTHESIS
4.0 400
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BLACk MUSCAT
" AQU€QUS PHASE
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f:I:IraljE; ~.~, ~.: ../ J h ,_" :. " .R<f 0·1, TO .' 0 FQR ALL STAGES
8 9 .. 0
WEE KS AFTER ... ,..THE$JS
JVLS)(I MUSCAT
BLACK MUSCAT
Fig. 3. Endogenous level of cytokinins in seeds at various stages
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S.N. PANOEY AND kANjlT SINOH
The level of auxins further increased during the 7th week to 42 % in Black Muscat and 32% in Julski Muscat at Rf 0.3 and to 37% at Rf 0.3 in the former cultivar and 25% at Rf 0.2 in the latter during the 8th week. The data show that the same type of auxins did not remain the most acHve one during all stages. The most active Rf. zone was 0,6 in the 3rd week, 0.9 in the 4th, 0.3 during the 7th and 8th weeks in Black Muscat. In Julski Muscat, it was 0.4 in the 3rd week, 0.3 during the 4th and 7th weeks and 0.2 in the 8th week. Towards the harvesting time the level of aU1l.ins decreased drastically, 17% during the 9th, 9% during the 10th and 4% during the 11th week in Black Muscat, and 12% during the 9th week in Jutski Muscat. The maximum decrease was observed at Rf0.8 during the 11th week in the former cultivar and at Rf 0.9 (9%) in the latter cultivar.
Gibberellin-like substances
Black Muscat and Julski Muscat behaved differently with regard to the level of gibberellin-like substances (Fig. 2). It is seen that the gibberellins at certain Rf zones actually showed inhibitory effect.
At the end of the 3rd week, gibberellins showed high activity (about 70%) at RfO.7, 0.8 and 1.0 in Black Muscat and 0.8 in Julski Muscat. Whereas the level of gibberellins increased to 97.5 % at Rf 0.1 in the former cultivar during the 4th week, it decreased maximum to 48% at Rf 0.2 in the latter cultivar at this
stage.
None of the Rf zones showed promoting effects in Black Muscat during the 5th and 6th weeks but the level increased to 32.40% at Rf 0.8 during the 7th week. On the oth«::r hand, quite a high level of gibberellins was observed in Julski Muscat at Rf 0.8 (33%) during the 5th week which increased to 49% during the 6th week at the same Rf and 52.50% at Rf 0.9 during the 7th week. The level of gibberellins then decreased to 25 % in Black Muscat and 33 % in Julski Muscat during the 8th week, which decreased progressively alongwith the advancement in maturity reaching to traces at the ripening of berry in both cultivars.
Cytokioins
A very high level of cytokinins was observed during the first two stages in both cultivars (Fig. 3). Thererfter, kinin-like activity disappeared completely during the 5th week. Rather all Rf zones at the later stages were found to be inhi
bitory. However, the two cultivars behaved differently with regard to the nature of cytokinins and their levels present in seeds during the initial two stages. The water soluble cytokinins dominated in Black Muscat during the 3rd week at Rf
HORMONES IN DEVELOPING GRAPIl SEED 303
cytokinins exhibited very strong activity in Iulski Muscat at Rf 0.9 (720%) during the 3rd week. The level of cytokinins, in general, was higher in early maturing cv. Iulski Muscat than the other cultivar.
Inhibitors
The inhibitors were present in seeds right from the beginning and were found to increase progressively in both cultivars (Fig. 4). It may be noted that the most active Rf zone shifted during the various stages and the inhibitors other than absci sic acid (ABA) were also present at the various Rf zones.
In Black Muscat, the maximum activity was found at Rf 0.7 during the 7th week and at 0.6 (ABA-like substances) during the 8th week, whereas the maximum activity was at Rf 0.8 from the 9th to 11th weeks.
The lowest level of inhibitors was observed during the 3rd week in both culti vars. It may also be seen that the highest level of inhibitors observed in Black Muscat (12%) and Iulski Muscat (14%) at Rf 0.8 dUling the 3rd week continued to remain the same in the following week in both cultivars but the maximum active Rf zone shifted to 0.7 in the former cultivar. During the 5th week, the inhibitors started building up substantially at all Rf zones in both cultivars leading to the maximum increased level at Rf 0.4 and 0.5 in Black Muscat (20%) and at Rfo.5 in Iulski Muscat (22%). lhe level increased further during the 6th week in both cultivars at all but Rf 0.4 in Black Muscat. However, the most active Rf zone during this period was 0.8 in Black Muscat (28%) and 0.5 in Iulski Muscat (24%). The inhibitors increased progressively during the growth and develop ment of seeds, reaching the maximum at the ripening of berries.
From the above, it is obvious that the level of auxin, gibberellin and cytokinin like substances, varied as the seeds matured. Two distinct phases were noted when the levels of the above growth regulators were relatively higher. The lag phase was noticed during the 5th and 6th weeks.
Similar activity of gibberellin-like substances was reported during the lag phase in cv. Perlette (Bhullar and Dhillon, 1974). Contrarily, cytokinin disappeared completely with the onset of the lag-phase in seeds during the 5th week in the present study, which is in conformity with Gazit and Blumenfed (1970) and Oritani and Yoshida (1971), who reported a high cytokinin activity in developing fruits and seeds and noticed falling level as the tissues matured making it difficult to detect. Inhibitors in -the present study incre~ed ~adually froJ'll the 3rd wee~ to the ripen.
S.N. PANDEY AND ltANJIT SINGR
fa
High level of growth promoting substances and low level of inhibitors might be responsible fOr rapid growth of seeds as well as that of berries (Rao and Pandey, 1976) during the initial stages of growth. The absence of gibberellins, low level of auxins and high level of inhibitors in seeds during the 5th and 6th weeks have obviously not been conducive to growth of seeds in Black Muscat inducing a lag phase in seed growth. This appears to have necessitated the arplication of gibbere llic acid and kinetin for getting seeds germinated without stratification 5 weeks after anthesis (Pandey, 1982; Pandey and Singh, 1987, 1988). However, the seeds of Julski Muscat in the present inve!<tigation showed higher gibbere1lins than Black Muscat during the lag-phase. The higb level of inhibitors in the seeds during post lag phase (7th and 8th weeks) in the present study, did not anow seeds to show distinct growth (Pandey, 1982).
ACKNOWLEDGEMENT
Senior author is grateful to Dr. R.M. Pandey, then Head of the Division of Fruits and Horticultural Technology, Indian Agricultural Research Institute, New Delhi for his keen interest and kind technical advice.
REFERENCES
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Vilis vil'/i/era L.) Fiziologiya na Rasteniyala, 6 (2) : 70-76.
Bhullar, J.S. and Dhillon, B.S. (1974). Natural growth substances in Perlette grares in relation to berry development. Haryana J. Horl. Sci., 3 : 106-12.
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Anab-e-Shahi grapes. Vilis, 15{l) : 5-10.
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Oazit, S. and Blumenfeld, A. (1970). Cytokinin and inhibitor activities in the Avocado fruit
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Letham. D.S. (1968). A new cytokinin bioassay and tbe naturally occurring cytokinin complex.
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305
HORMONES IN DEVELOPING GRAPE SEED
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