Fruit maturity, storage and postharvest maturation treatments affect bell pepper
( Capsicum annuum L. ) seed quality*
Vic tor M. Sanchez **,l, F.J. S u n d s t r o m 1, G .N . M c C l u r e a n d N.S. Lang
Department of Horticulture, Louisiana Agricultural Experiment Station, LSU Agricultural Center, Baton Rouge, LA 70803, USA
(Accepted 4 February 1993)
ABSTRACT
Some peppers (Capsicum sp. ) exhibit a primary seed dormancy at harvest. This study was designed to examine the effect of fruit maturity, dry and moist seed storage and seed postharvest maturation treatments on the seed germination of four bell pepper cultivars (Capsicum annuum L., cultivars 'Early Calwonder', 'Resistant Giant No. 4', 'VR2' and 'Yolo Wonder'). In 1987 and 1988, fruits were harvested 30 (mature green), 40 (breaker), 50 (mature red) and 60 (over-mature red) days post- anthesis (dpa), and seeds were extracted for dry storage treatments, or allowed to remain in fruits for postharvest maturation treatments of 0, 7, 14, 21 and 28 day periods. In 1988, seeds were also stored over water or saturated salt solutions which provided relative humidities of 7, 51 and 97.5% to deter- mine the influence of seed moisture during seed storage. Seeds from red and over-mature red ( 50 dpa and 60 dpa, respectively) fruit generally had greater dry weights and higher germination percentages relative to seeds from less mature fruits. Seeds extracted from mature green (30 dpa) fruit did not germinate regardless of storage time. Seed postharvest maturation of up to 14 days in green fruit significantly increased seed germination in all cultivars. When pepper seeds were allowed to remain in mature red (50 dpa) fruit for various postharvest maturation periods, germination percentages were generally significantly greater than in dry storage and mature green ( 30 dpa) postharvest matur- ation treatments. Starch concentrations of 'Resistant Giant No. 4' seeds increased from 10 to 80 mg glucose per 100 g dry wt. when seeds remained in fruit for 4 weeks after harvest. Dry storage of seeds following extraction from red fruit did not significantly increase germination, indicating that seed aflerripening did not occur. Short storage periods (7 and 14 days) at low relative humidity (7 and 51% ) significantly improved seed germination of'Early Calwonder' and 'Resistant Giant No. 4'. Stor- age for 7-14 days of 'Early Calwonder' and 'Resistant Giant No. 4' seeds at high (97 and 100%) relative humidities was also beneficial, but at longer periods germination percentages of all cultivars dropped. There were few significant differences in seed dry weight between dry storage and posthar- vest maturation treatments. It is possible that in situ priming may have occurred in 30 and 50 dpa seed postharvest maturation treatments. These results suggest that following fruit harvest, pepper seeds should remain in red (50 dpa) fruit for a short postharvest maturation period to achieve max- imum seed germination potential.
*Paper no. 91-28-5296 of the Journal Series, Louisiana Agricultural Experiment Station. **Corresponding author. 'Present address: INCOTEC, 1293 Harkins Road, Salinas, CA 93901, USA.
Bell pepper seeds often exhibit dormancy at harvest (Randle and Honma, 1981 ), and for maximum germination require a period of dry storage follow- ing harvest referred to as afterripening (Odland, 1938; Randle and Honma, 1981; Bewley and Black, 1982; Edwards and Sundstrom, 1987 ). Pepper seed germination has also been increased by allowing seeds to remain in harvested immature fruits for up to 30 days prior to extraction (Cochran, 1943 ). High quality Capsicum seed can also be obtained from mature red fruits (Duczmal and Kaczmarkiewicz, 1984). Both fruit maturation and dry afterripening contributed to overcoming seed dormancy in various Capsicum species (Randle and Honma, 1981; Edwards and Sundstrom, 1987 ).
Seed moisture has been demonstrated to play an important role in the per- sistence of dormancy of some crucifer seeds. With some crops such as rape (Brassica rapus), seed desiccation accelerated the removal of dormancy; yet with other crops such as leaf mustard (Brassica cernua) and potherb mustard (Brassica japonica), the removal of seed dormancy was suppressed by desic- cation (Tokumasu et al., 1981 ). Seed storage under high relative humidity conditions (50-60%) can improve the germination percentage of some weed seeds (Baskin and Baskin, 1979).
This work was undertaken to determine the effect of fruit maturity, seed storage at low and high moisture, and postharvest fruit maturation on the seed germination characteristics of four bell pepper cultivars (Capsicum an- nuum L., cultivars 'Early Calwonder', 'Resistant Giant No. 4', 'VR2' and 'Yolo Wonder' ).
MATERIALS AND METHODS
Plant material - The seeds of the open-pollinated bell pepper cultivars 'Early Calwonder', 'Resistant Giant No. 4', 'VR2', and 'Yolo Wonder' were seeded in a soilless media in Todd 100A Planter fiats (Speedling, Sun City, FL) for transplant production in the greenhouse during the spring of 1987, 1988, and 1989. After 6 weeks, seedlings were hardened (by lowering temperature and soil moisture ) and transplanted in the field. Each cultivar was isolated from the others by 15 m planted in corn to reduce undesired cross-pollination. The soil was an Olivier silt loam (fine-silty, mixed, thermic, Aquic, Fragiudalf) with 0.60% organic matter and a pH of 6.3. All plots received a preplant band application of N - P - K (8-0.1-0.2) fertilizer at a rate of 636 kg ha -1 10-12
V.M. Sanchez et al./Scientia Hortic. 54 (1993) 191-201 193
cm under the row center, and 67 kg ha- ~ of ammonium nitrate (NH4NO3) as a sidedressing 3 weeks following transplanting and immediately after fruit set. Plant in-row spacing was 30 cm on rows spaced 1.2 m apart and there were 16 plants per plot. Plots of each cultivar were arranged in a completely randomized design with six replications. All flowers of all plants were tagged at anthesis for determination of fruit maturity.
Fruit maturity study. - In 1987 and 1988, fruits of each cultivar were har- vested at 30 days, 40 days, 50 days and 60 days post-anthesis (dpa) corre- sponding to mature green, breaker, mature red and over-mature red stages, respectively. From each harvest, ten fruits per cultivar were selected ran- domly from each plot and seeds were manually extracted and weighed. Seeds were surface-dried for 24 h at 25 °C before germination testing.
Dry storage and postharvest maturation studies. - During the 1987 and 1988 fruit maturity studies, additional fruits from both the 30 and 50 dpa harvests were collected. Seeds were either extracted from fruit and placed within dry storage treatments, or were stored within intact fruits for postharvest matur- ation treatments. Prior to storage, all fruits were dipped for approximately 1 min in a 10% NaOC1 solution to retard fruit decay. Both treatments were performed at 25 °C in dark incubators and seeds in each treatment were stored for 0, 7, 14, 21, and 28 day periods. Each week during this 4 week period, seeds were extracted from randomly chosen fruits in postharvest maturation treatments, or collected from dry storage treatments for weighing and germi- nation testing.
A second study was initiated in 1988 to investigate the effects of moisture during the storage of seeds extracted from fruits harvested at 50 dpa. Various seed moisture contents were achieved by equilibration in sealed containers at 25 °C over three saturated salt solutions, NaOH, Ca (NO3)2, K2SO4, and water, with relative humidities of 7%, 51%, 97.5% and 100%, respectively (Winston and Bates, 1960). Extracted seeds were only surface-dried before they were placed over solutions to avoid any desiccation-triggered germinative events that would not occur in seeds stored in fruits at high moisture (Bewley et al., 1989). Seeds were stored in a dark incubator for a 28 day period and were removed every 7 days for germination, dry weight and moisture percentage determination as previously described (Sundstrom, 1990).
A third study was initiated in the spring of 1989 to contrast the differences in seed dry weight and stored carbohydrates between dry storage and posthar- vest maturation treatments. 'Resistant Giant No. 4' fruit were harvested at 50 dpa, and intact fruit and extracted seeds were stored at 25 °C as previously described. As before, every 7 days for a 28 day period seeds were collected from dry storage, or extracted from stored fruit for sizing and weighing. To avoid the large variability in seed size between different fruits, only seeds
194 V.M. Sanchez et al./Scientia Hortic. 54 (1993) 191-201
passing through a 4 mm but not a 3 mm mesh USDA screen were used for measurements.
Seeds from each treatment were then ground with a 20 mesh Wiley mill, weighed, and non-structural, soluble carbohydrates were extracted in 80% (v/ v) ethanol from 200 mg samples. Concentrations of glucose, fructose, sucrose and maltose were determined using the high performance liquid chromato- graphy (HPLC) method as described by Robbins and Pharr (1988), with minor modifications which included acetonitrile at a flow rate of 1.0 ml min- as the solvent. Sugars were identified using an Aminex HPX-87C column (Bio- Rad Laboratories, Richmond, CA). Starch concentrations were determined enzymatically by detecting released glucose (Robbins and Pharr, 1988 ). Ow- ing to limited seed supplies, composite samples of each treatment combina- tion were chemically analyzed.
S e e d qual i ty tests. - Germination tests were conducted at 25 ° C in the dark, and consisted of eight replicates of 50 seeds placed on one layer of Whatman No. 2 filter paper moistened with 4 ml of distilled water in 90-ram Petri dishes. Germination counts were performed daily under light for 15 days. Seeds were considered to have germinated at the first sign of radicle protrusion. The mean time to germination (MTG) was calculated as
MTG--- ( T N + T2N2 + ... + TnNn) / X
where T is the day number, N is the number of seeds germinated that day and X is the total number of seeds germinated. The germination percentage and mean number of days for radicle emergence were calculated as described by Edwards and Sundstrom (1987). Seed dry weight was measured following drying at 75°C for 24 h.
Stat is t ical analysis. - Germination percentage and MTG data were arcsine and log transformed, respectively, prior to analysis using the Statistical Anal- ysis System (SAS, 1985) GLM procedure. Only non-transformed data are presented for ease of interpretation in tables and figures. Mean separations were determined for data from the fruit maturity experiment utilizing LSD(0.05). Differences between all possible treatment combinations were examined (GLM procedure; SAS, 1985 ) for the dry storage and postharvest maturation studies, as well as the experiments on seed storage at low and high relative humidities.
RESULTS AND DISCUSSION
Frui t m a t u r i t y study. - Cultivar X maturity × year, and cultivar X maturity in- teracted significantly (P < 0.01 ) in their effect on germination percentage and rate. We attribute significant year interactions to large growing season rainfall
differences between the 2 years. Because of these interactions, 1987 and 1988 germination data are presented separately in Table l, and only 1988 repre- sentative data are presented in Fig. 1. MTG data for 40 dpa fruit are not included in Table l and Fig. l because total germination percentages were too low to provide meaningful estimates of MTG. As fruit maturity increased, seed germination percentages of all cultivars increased (Table 1 ). Seeds ex- tracted from mature green fruit (30 dpa) did not germinate. In all cultivars, seeds extracted from mature or over-mature red fruits (50 dpa and 60 dpa, respectively) had significantly higher germination percentages than seeds from fruits harvested at 40 dpa (breaker stage). There were generally no significant differences in MTG between 50 and 60 dpa harvests (Table 1 ). As fruit ma-
T A B L E l
Influence of fruit m a t u r i t y o n average seed dry weight and moisture, mean time to germination ( M T G ) ,
and percentage germination
D a y s p o s t - A v g . seed Seed 1 9 8 7 1 9 8 8
anthesis j d r y wt . 2 moisture 2 ( m g ) ( % f r e s h wt . ) M T G 3 Germination MTG 3 Germination
( d a y s ) ( % ) ( d a y s ) ( % )
'Early Calwonder" 30 2 .9 c 72 .6 ~ . . . .
4 0 3 .9 b 59 .8 b - 3 ¢ - 16 b
50 5 .9" 4 3 . 8 ¢ 6 .0" 4 3 b 6 .1" 81 a
6 0 6 .0" 4 5 . 9 c 7 .3 b 92" 7.3 b 92"
'Resistant Giant No. 4" 30 2 .5 c 77.1 a - - -
4 0 5 .9 b 5 3 . 3 b - 1 ~ - 8 ~
5 0 7 .6" 4 6 . 7 ¢ 6 .3" 25 b 5 .4 a 67 b
6 0 6 .2 b 4 7 . 8 c 6 .3" 93 a 6 .4 a 89 ~
'VR2' 3 0 3.'3 ¢ 7 3 . 5 " - - - -
4 0 6 .4 b 51 .8 b - 15 c - 2 b
50 7 .2 a 4 7 . 5 c 5 .9 a 52 b 5.3 a 55"
6 0 6 .7 ~b 4 9 . 3 c 5 .3 ~ 8 4 a 4 .7 a 4 9 a
'Yolo Wonder ' 3 0 2 .5 d 7 5 . 7 " - - - -
4 0 5.3 c 53. I b - 13 c - 4 c
50 6 .4 b 5 0 . 9 b 7 .2 a 4 9 b 5 .4 a 76 b
6 0 7 .5" 50 .5 b 5 .4" 88 a 5 .4 a 9 4 a
I Days post-anthesis (dpa): 30, mature green; 40, breaker; 50, mature red; 60, over-mature red. 2Measured only in 1988 .
3Data for 40 dpa not calculated because germination percentages were t o o l o w t o provide meaningful estimates. Means separated by cultivar within columns b y L S D at the 5% level.
! 96 V.M. Sanchez et al./Scientia Hortic. 54 (1993) 191-201
IL M. Sanchez et al. /Scientia Hortic. 54 (1993) 191-201 197
turity increased, seed moisture decreased from an average of 75% at 30 dpa to 26% at 60 dpa. With the exception of 'Yolo Wonder', there was not a clear relationship between maximum seed germination percentage and seed phys- iological maturity (maximum seed dry weight). Welbaum and Bradford (1989) found that major increases in muskmelon (Cucumis melo L. ) seed vigor occurred after maximum seed dry weight had been achieved. This sug- gested that further pepper seed maturation within the fruits might be benefi- cial to achieve maximum germination potential.
Dry storage and postharvest maturation studies. - There was a significant three- way interaction between cultivar, storage treatment (dry storage or posthar- vest maturation), and storage duration. There was a significant (P< 0.01 ) two-way interaction between storage treatment and storage duration for both germination percentage and rate for each cultivar. Therefore, data are pre- sented separately by cultivar in a series of graphs illustrating this interaction (Fig. 1 ). Seeds extracted from fruits harvested 30 dpa did not germinate re- gardless of storage time (data not shown). The germination percentage of seeds extracted from fruits harvested at 50 dpa and stored for a 28 day period did not significantly improve over time (Fig. 1 ). These observations indicate that dry afterripening did not occur during dry storage in any of the four cul- tivars tested, and are in contrast to previous results with Capsicumfrustescens seed (Edwards and Sundstrom, 1987). Randle and Honma (1981) found that some, but not all, cultivars of C. annuum responded to seed dry afterripening.
Germination percentages of all cultivars increased significantly (P= 0.001- 0.0166 ) when seeds were allowed to remain in mature green (30 dpa) fruit for a 14 day postharvest maturation period (Fig. 1 ). The germination per- centages of'Early Calwonder' and 'Resistant Giant No. 4' seeds continued to increase significantly (P= 0.0001 and P = 0.0019, respectively) when they re- mained for up to 21 days in mature green fruit.
When bell pepper seeds were allowed to remain in mature red (50 dpa) fruits for specific postharvest maturation periods, with the exception of'Early Calwonder' at 28 days (P=0.0560) and 'Yolo Wonder' at 7, 21, and 28 days, germination percentages were significantly (P= 0.0001-0.0076 ) greater than in other treatments (dry storage and postharvest maturation of seeds from 30
Fig. 1. The effect of fruit maturity, dry storage and postharvest maturation treatment time on seed germination percentage and rate (MTG) of'Early Calwonder' (A and E), 'Resistant Giant No. 4' (B and F), 'VR2' (C and G), and 'Yolo Wonder' (D and H). Treatment designated by: A, days in green fruit; I1, days out of mature fruit; 0 , days in mature red fruit. Error bars indi- cate standard errors for a mean of eight replicates of 50 seeds each at each sampling date. Error bars are not depicted if they are smaller than the symbols used to designate mean values.
198 KM. Sanchez et al./Scientia Hortic. 54 (1993) 191-201
dpa fruit, and dry storage of seeds from 50 dpa fruit). After only 7 days of in- fruit storage, germination of 'VR2' pepper seeds increased from 55 to 88%, eventually increasing to 96% after 28 days. In contrast to these laboratory findings, germination of 'VR2' did not increase between 50 and 60 dpa field harvests (Table 1 ). With the exception of the 7-day seed storage period of 'Early Calwonder', 'Resistant Giant No. 4', and 'Yolo Wonder' (P=0.0616, P= 0.5707, and P = 0.6495, respectively), the MTG of seeds stored in red (50 dpa) fruit was significantly (P=0.0001-0.0025) lower than 50 dpa out-of- fruit treatments (Fig. 1 ). Similar results with pimento pepper were reported by Cochran ( 1943 ).
There were no significant differences in seed dry weights between dry stor- age and postharvest maturation treatments (data not shown). Differences in moisture percentages between seeds in postharvest maturation and dry stor- age treatments were dramatic. After 28 days, the mean cultivar moisture per- centage of seeds which remained in fruit was 40.2, vs. 4.1 for seeds in dry storage. To test the influence of seed moisture during storage on germination, seeds were treated with fungicide and equilibrated over saturated salts or water. After 28 days, the mean seed moisture percentages over NaOH, Ca(NO3)2, K2SO4 and water were 4.2, 9.1, 33.5 and 36, respectively.
Cultivar, relative humidity (RH), and storage treatments significantly (P< 0.01 ) interacted in their effect on germination percentage and rate, thus cultivar data have been presented separately. The interaction of RH and stor- age time on germination percentage was significant (P< 0.05 ) for 'Early Cal- wonder' and highly significant (P<0.01) for the remaining cultivars (Fig. 2). The effect of moisture on seed germination was not consistent within each cultivar. Seven and 14 days storage increased significantly seed germination of 'Early Calwonder' and 'Resistant Giant No. 4' (P= 0.0008-0.0168 and P= 0.0006-0.0151, respectively), in comparison with no storage, irrespective of RH (Fig. 2). Storage at high RH for longer periods, however, decreased the germination of those two cultivars. When seeds of 'VR2' and 'Yolo Won- der' were stored at 97 and 100% RH for more than 14 days, there was a sig- nificant (P= 0.0001 ) deleterious effect on germination (Fig. 2 ). 'VR2' seed germination was low regardless of RH treatment, indicating that only pos- tharvest maturation periods within fruits were successful in increasing ger- mination percentages. It was not possible to raise the moisture content of seeds extracted from fruits to levels comparable to those of seeds stored within fruits.
Fig. 2. The effect of the two-way interaction of storage time and RH on seed germination per- centage of: (A) 'Early Calwonder'; (B) 'Resistant Giant No. 4'; (C) 'VR2'; (D) 'Yolo Won- der'. Each bar represents the mean of eight replicates of 50 seeds each. Mean standard errors for each cultivar for relative humidities of 7%, 51%, 98% and 100% are respectively: (A) 1.4, 1.6, 3.9, 2.9; (B) 3.8, 3.4, 4.5, 4.0; (C) 4.8, 6.7, 7.6, 3.7; (D) 3.6, 3.2, 0.7, 3.7.
V.M. Sanchez et al./Scientia Hortic. 54 (1993.) 191-201 199
GERMINATION (%)
100 I 9O 80 70 60 EO 4O 3O 2O ' " V ~ / ~ - o /
0 7 14 21 28
GERMINATION (%) g
lOO
8o 7o EO 60 40 1 )0 30 97.6 20 lO ) RH
0 0 7 14 21 28
O
(%) RH
GERMINATION (9=)
0 7 14 21 28
GERMINATION (.i;)
100 90 80 70 60 50 40 30 20 10 0
0
D
97.5
) RH
14 21 28
S T O R A G E (DAYS)
200 V.M. Sanchez et al./Scientia Hortic. 54 (1993) 191-201
Mean water content of seeds harvested from fruits of all cultivars at 60 dpa (roughly equivalent to one-third of the way through the postharvest matura- tion study) was 48.4% (Table 1 ). After 28 days in the postharvest maturation study, seed moisture was approximately 40%. This implies that for much of the storage experiment, seeds within the fruit had a moisture content about 25% greater than the highest moisture content of the seeds stored over water (which at 28 days was 36%). Although there was not a clear indication of seed germination enhancement outside the fruit at a moisture content of 36%, seeds at higher moisture levels within pepper fruits may have been 'primed' in situ during the postharvest maturation period (Welbaum and Bradford, 1991).
There were no significant differences in seed glucose, fructose, sucrose or maltose concentrations between 'Resistant Giant No. 4' seeds in dry storage and postharvest maturation treatments (data not shown ). The starch concen- tration of seeds that were held in dry storage for up to 28 days remained rel- atively stable, varying between 10 and 19 mg glucose per 100 g dry wt. After 14 days, seeds stored within mature red (50 dpa) fruit had substantially in- creased starch concentrations compared with those in dry storage treatments (65 vs. 19 mg glucose per 100 g dry wt. at 21 days, and 80 vs. 19 mg glucose per 100 g dry wt. at 28 days). This increase in seed starch reserves, however, did not correspond well with germination data in Fig. 1.
The positive effect on germination of seed postharvest maturation periods in mature green (30 dpa) and mature red (50 dpa) fruit may be related to in situ priming (Welbaum and Bradford, 1991 ). The enhancement of germina- tion percentages of less mature seeds (harvested 30 dpa), may also be related to physiological maturation, and in mature seeds (harvested 50 dpa), meta- bolic advancement (priming) may be occurring. Consequently, the results of these studies suggest that following fruit harvest, pepper seeds should remain in red (50 dpa) fruit for a short postharvest maturation period to achieve maximum seed germination potential.
ACKNOWLEDGMENT
We gratefully acknowledge the assistance of R. G. Paul Clifton and Rebecca Frederick in the collection and analysis, respectively, of these data. We also thank Mayela Sanchez for her contributions throughout this project.
REFERENCES
Baskin, J.M. and Baskin, C.C., 1979. Effects of relative humidity on afterripening and viability in seeds of the winter annual Draba verna. Bot. Gaz., 140: 284-287.
Bewley, J.D. and Black, M., 1982. Physiology and Biochemistry of Seeds in Relation to Germi- nation. Vol. 2. Viability, Dormancy and Environmental Control. Springer, New York, 306 PP.
I(M. Sanchez et al./Scientia Hortic. 54 (1993) 191-201 201
Bewley, J.D., Kermode, A.R. and Misra, S., 1989. Desiccation and minimal drying treatment of seeds of castor bean and Phaseolus vulgaris which terminate development and promote germination cause changes in protein and messenger RNA synthesis. Ann. Bot., 63: 3-17.
Cochran, H.L., 1943. Effect of stage of fruit maturity at time of harvest and method of drying on the germination of Pimento seed. Proc. Am. Soc. Hortic. Sci., 33:477-480.
Duczmal, K.W. and Kaczmarkiewicz, M., 1984. Effect of planting time and harvesting method on Capsicum seed yield and quality. Hortic. Abstr., 55:5345; 1985.
Edwards, R.L. and Sundstrom, F.J., 1987. Afterripening and harvesting effects on Tabasco pep- per seed germination performance. HortScience, 22:473-475.
Odland, M.L., 1938. Observations on dormancy in vegetable seed. Proc. Am. Soc. Hortic. Sci., 35:562-565.
Randle, W.M. and Honma, S., 1981. Dormancy in peppers. Sci. Hortic., 14:19-25. Robbins, N.S. and Pharr, D.M., 1988. Effect of restricted root growth on carbohydrate metab-
olism and whole plant growth of Cucumis sativus L. Plant Physiol., 87:409-413. Statistical Analysis Institute, 1985. Users Guide: Statistics. SAS Institute, Cary, NC. Sundstrom, F.J., 1990. Seed moisture influences in Tabasco seed viability, vigor, and dormancy
during storage. Seed Sci. Technol., 18:179-185. Tokumasu, S., Kakihara, F. and Kaio, M., 1981. The change of dormancy of seeds stored in
desiccators and/or harvested fruits in cruciferous crops. Jpn. Soc. Hortic. Sci., 50:208-211. Welbaum, G.E. and Bradford, K.J., 1989. Water relations of seed development and germination
in muskmelon (Cucumis melo L. ). II. Development of germinability, vigor and desiccation tolerance. J. Exp. Bot., 40:1355-1362.
Welbaum, G.E. and Bradford, K.J., 1991. Water relations of seed development and germination in muskmelon (Cucumis melo L.). IV, Influence of priming on germination responses to temperature and water potential during seed development. J. Exp. Bot., 42:393-399.
Winston, F.P.W. and Bates, D.H., 1960. Saturated solutions for the control of humidity in bio- logical research. Ecology, 41:232-237.
