(NaCl) SPRAYS TO OVERCOME THE SELF ...

RESEARCH ARTICLE

SABRAO Journal of Breeding and Genetics 44 (2) 339-348, 2012

EFFECT OF COMMON SALT (NaCl) SPRAYS TO OVERCOME THE SELF-INCOMPATIBILITY IN THE S-ALLELE LINES OF Brassica oleracea var. capitata L. 1

SAURABH SINGH1* and VIDYASAGAR1

Department of Vegetable Science and Floriculture, CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur, H.P., India – 176062 *Corresponding author’s E-mail: [email protected]

SUMMARY Among the various methods for the maintenance of S-allele parental lines, the use of NaCl solution sprays has been reported as the most convenient and economic. The present investigation deals with the objective of ascertaining the concentration and number of sprays of common salt (NaCl) effective for temporary breakdown of selfincompatibility in the S-allele lines (1-4-6 and II-12-4-7) of cabbage. The S-allele lines were grown at the Experimental Farm and the pollinations viz. BP (selfing in bud stage) and OP (selfing in freshly opened flowers) were carried out manually in the plants enclosed with insect proof nylon nets. There was seed-set in BP on all the test plants during 2009-2010, but erratic seed-set in OP after NaCl sprays in the S-allele line I-4-6 probably due to too long time lag between spray and pollination and also the use of relatively lesser volume of spray (25 ml/plant). However, in II-12-4-7, there was practically no seed-set in OP probably due to the presence of stronger S-allele. The results obtained during second experimental year (2010-2011) proved that NaCl sprays (3% and 5%) are effective in temporary breakdown of self-incompatibility. Both the concentrations of NaCl spray were at par in the S-allele line I-4-6 whereas, 5% concentration was significantly superior in the S-allele line II-12-4-7 for seed-set in OP. Seed-set in OP across flowering regimes were at par in the both S-allele lines. All the three times of NaCl spray were at par for seed-set in OP in the S-allele line I-4-6 whereas, NaCl sprays made 15 minutes after and 15 minutes before plus after pollination were significantly superior to common salt sprays made 15 minutes before pollination in II-12-4-7. Keywords: Cabbage, self-incompatibility, common salt (NaCl), seed production, open pollination, bud pollination Manuscript received: February 28, 2012; Decision on manuscript: August 28, 2012; Manuscript accepted in revised form: October 17, 2012. Communicating Editor: Bertrand Collard

SABRAO J. Breed. Genet. 44 (2) 339-348, 2012

INTRODUCTION

MATERIALS AND METHODS

Cabbage (Brassica oleracea var. capitata L.) is one of the most important Cole crops being grown throughout the world and has originated from wild cabbage (Brassica oleracea var. oleracea L.). The genetic mechanism of sporophytic self-incompatibility has proved effective in hybrid seed production of Cole crops. For continuous production of hybrid seeds, maintenance of parental selfincompatible (SI) lines is one of the basic requirements. Earlier, the seeds of S-allele lines were produced by manual sib mating in bud stage (Cabin et al., 1996). However, bud pollination is time consuming and suitable for the production of small seed quantity only. Similarly, exposing plants to high CO2 concentration (3-5%) and relative humidity (100%) in air tight growth chamber for production of seeds of parental S-allele lines (Palloix et al., 1985) also require costly infrastructure. Off late, a new, inexpensive and easy to use method was given by Liao (1995), in which sodium chloride solution was used to breakdown the selfincompatibility in Brassica spp. including cabbage. However, the use of sodium chloride (NaCl) solution in Cole crops has not yet been investigated in India. Hence the present investigation was undertaken with the objective of ascertaining the concentration and number of sprays of common salt for temporary breakdown of self-incompatibility in the S-allele lines of cabbage.

The investigation was undertaken at the Experimental Farm of the Department of Vegetable Science and Floriculture during 2009-2011. The S-allele lines of cabbage i.e. I-46 and II-12-4-7 were used to study the effect of common salt (NaCl) solution in temporary breakdown of self-incompatibility. The experiment was conducted for two years, 20092010 and 2010-2011. During 20092010, the effect of NaCl solution sprays in overcoming the selfincompatibility was observed after 24 hours to 96 hours of spray as reported by earlier researchers up to 120 hours of spray. Based upon the first year results (2009-2010), the effect of NaCl solution sprays in breakdown of self-incompatibility was observed 15 minutes before pollination and 15 minutes after pollination during 2010-2011 as per the findings of Liao (1995). During 2009-2010 seeds of S-allele lines of cabbage were sown in the nursery on 10th September, 2009 and transplanting of seedlings was carried out on 20th October, 2009 at spacing of 60 x 45 cm in open field conditions. For the year 2010-2011, nursery sowing of the seeds of Sallele lines, produced through selfing in bud stage and sib mating in control plants, was done on 1st September, 2010 and transplanting was carried out on 13th October, 2010 at 60 x 45 cm spacing. All the recommended package of practices was followed to ensure the proper growth of plants. Cross cuts to heads were given from 2nd fortnight of February to early March to hasten bolting. The plants of self incompatible lines were enclosed with the insect proof nylon net

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Singh & Vidyasagar (2012)

RESULTS

enclosures to prevent out crossing by pollinators. The concentrations of common salt (3% and 5%) and control treatment (no spray) were same in both the experimental years. Three plants per treatment and 15-20 flowers/buds/pollination were taken. During the first year (20092010), the spray quantity of NaCl solution was 25 ml/plant/spray and three sprays of NaCl solution were made. The first spray was made at 15-20 per cent flowering and subsequent sprays were made after every four days. The pollinations OP (selfing in freshly opened flowers) and BP (selfing in bud stage) were started from one day after spray (1DAS) up to 4DAS. The S-allele lines were also maintained through manual sib mating in bud stage. For the second experimental year (20102011), spray quantity of NaCl solution increased to 50 ml/plant/spray. The spray of common salt (NaCl) solution was made 15 minutes before, 15 minutes after and 15 minutes before plus after OP (selfing in freshly opened flowers) and BP (selfing in bud stage) pollinations during the three flowering regimes i.e. 25-50%, 5075% and >75% flowering. Average number of seeds per siliqua in each treatment were counted and recorded. Data were analysed as suggested by Panse and Sukhatme (1985).

In the first year (2009-2010) the average number of seeds per siliqua obtained as the mean of three plants in OP (selfing in freshly opened flowers) and BP (selfing in bud stage) treatments in the S-allele lines I-4-6 and II-12-4-7 after the common salt sprays (3% and 5%) are presented in tables 1 to 2. All the plants set seeds in BP (selfing in bud stage) treatment confirming the viability of male and female gametes of the test plants. With the one spray of 3% common salt (NaCl) solution in the S-allele line I-4-6, seed-set in OP (selfing in freshly opened flowers) treatment was recorded only when the pollinations were carried out one day after spray (0.56 seeds/siliqua). With two sprays of 3% NaCl solution seed-set in OP treatment was obtained when the pollinations were carried out two days after spray (0.16 seeds/siliqua), 3 days after spray (0.03 seeds/siliqua) and 4 days after spray (0.13 seeds/siliqua). No seed-set in OP treatment was noted in any of the plants which had been given 3 sprays of common salt (Table 1). With one spray of 5 per cent common salt (NaCl) seed-set in OP treatment was recorded only when the pollinations were carried out one day after spray (0.10 seeds/siliqua). Similarly with two sprays of 5% NaCl solution some seed-set in OP treatment was obtained (0.43 seeds/siliqua) only when the pollinations were carried one day after spray. With three sprays of 5 % common salt no seed-set was obtained in OP treatment on any of the plants when the pollinations were carried out up to 4 days after spray. The control treatment (no

Statistical analysis Statistical analysis of experimental data was accomplished by Analysis of Variance in completely randomized block design (CRBD) using CPCS-1 software (Cheema and Singh, 1990).

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spray) was common for 3% and 5% common salt spray treatments. There was seed-set in BP treatments whereas no seed-set was obtained in OP treatments on the control plants (Table 1). The results obtained in the S-allele line II-12-4-7 during 20092010 with 3% and 5% common salt (NaCl) spray are presented in Table 2. All the plants produced seeds in BP treatment confirming the viability of male and female gametes of the test plants. No seed-set in OP treatment was recorded in any of the plants which had been given one spray, two sprays and three sprays of 3 per cent common salt (NaCl) solution when the pollinations were carried out up to 4 days after spray. With one spray of 5% NaCl solution in the S-allele line II-12-4-7, only 0.05 seeds/siliqua were obtained in OP treatment only when the pollinations were carried out one day after spray. No seed-set in OP treatment was noticed in any of the plants which had been given two sprays and three sprays of 5% NaCl solution (Table 2). There was seedset in BP treatments whereas no seed-set was obtained in OP treatments on the control plants (Table 2). During the experimental year 2010-2011, the average number of seeds/siliqua obtained as the mean of three plants in OP (selfing in freshly opened flowers) and BP (selfing in bud stage) treatments in the S-allele lines I-4-6 and II-12-4-7 after 3% and 5% common salt (NaCl) sprays and no spray (control) treatment are presented in Tables 3 to 4. All the plants set seeds in BP treatment confirming the viability of male and female gametes of the test plants. Good amount of seed-set was

obtained in OP treatment on all the test plants, across the flowering regimes (25-50%, 50-75% and >75% flowering), after the common salt (NaCl) sprays (3% and 5%) made 15 minutes before pollination, 15 minutes after pollination and 15 minutes before plus after pollination in the S-allele lines I-4-6 and II-124-7. However, no seed-set was recorded in OP treatments during each of the flowering regimes, on any of the plants in the control treatment (no spray) in both the Sallele lines I-4-6 (Table 3) and II-124-7 (Table 4) in which pollinations were carried out along with the plants sprayed with common salt solution. The effect on phenotype of NaCl sprays was also observed with respect to silique length. It was recorded that the length o silique in the plants treated with NaCl solution sprays was more and were well developed than the untreated plants. Hence the silique developed after salt sprays were having more number of seeds. However further studies are needed to pinpoint the effect at biochemical and molecular level.

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Table 1. Average number of seeds/siliqua in OP and BP after 3% and 5% spray of NaCl solution along with control (no spray) in the Sallele line I-4-6 (2009-2010). Conc. No. of sprays 1 2 3 NS(C)

3% 1 DAS OP 0.56 0.00 0.00 0.00

BP 4.96 2.11 3.27 3.18

2 DAS OP 0.00 0.16 0.00 0.00

5% 3 DAS

BP 1.38 2.71 1.67 0.56

OP 0.00 0.03 0.00 0.00

BP 1.34 3.16 1.51 1.27

4 DAS OP 0.00 0.13 0.00 0.00

BP 1.80 1.84 1.05 2.14

1 DAS OP 0.10 0.43 0.00 0.00

BP 1.97 1.05 1.55 3.18

2 DAS OP 0.00 0.00 0.00 0.00

3 DAS

BP 1.38 1.31 1.32 0.56

OP 0.00 0.00 0.00 0.00

BP 2.33 1.44 1.64 1.27

4 DAS OP 0.00 0.00 0.00 0.00

BP 2.48 1.14 2.11 2.14

Table 2. Average number of seeds/siliqua in OP and BP after 3% and 5% spray of NaCl solution along with control (no spray) in the Sallele line II-12-4-7 (2009-2010). Conc. No. of sprays 1 2 3 NS(C)

3% 1 DAS OP 0.00 0.00 0.00 0.00

BP 1.45 1.29 1.22 1.09

2 DAS OP 0.00 0.00 0.00 0.00

BP 1.52 1.75 1.25 1.34

5% 3 DAS OP 0.00 0.00 0.00 0.00

BP 1.68 1.34 1.40 1.18

4 DAS OP 0.00 0.00 0.00 0.00

OP – Self pollination in open flower stage, BP – Self pollination in bud stage DAS- Days after spray, NS – No spray (control)

BP 1.85 1.43 1.03 1.21

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1 DAS OP 0.05 0.00 0.00 0.00

BP 1.47 1.36 1.28 1.09

2 DAS OP 0.00 0.00 0.00 0.00

BP 2.21 1.50 1.11 1.34

3 DAS OP 0.00 0.00 0.00 0.00

BP 1.45 1.47 1.13 1.18

4 DAS OP 0.00 0.00 0.00 0.00

BP 2.35 1.40 1.16 1.21


Table 3. Average number of seeds/siliqua in OP and BP upon NaCl spray (3% and 5%) along with control (no spray) during different flowering regimes in the S-allele line I-4-6 (2010-2011). Conc.

3%

5%

Flowering Regimes

15 ( b ) OP BP

15 ( a ) OP BP

15 ( b + a ) OP BP

25-50% 50-75% >75%

1.63 4.86 4.15

3.23 5.33 1.40

5.12 3.54 3.31

4.27 5.05 6.14

4.38 5.83 2.66

6.97 4.43 3.26

NS ( C ) OP BP 0.00 0.00 0.00

2.27 2.79 1.76

15 ( b ) OP BP

15 ( a ) OP BP

15 ( b + a ) OP BP

NS ( C ) OP BP

3.50 1.68 1.33

2.63 7.55 3.38

3.56 3.94 3.91

0.00 0.00 0.00

3.06 2.41 4.11

5.25 6.94 4.23

2.93 4.12 3.57

2.13 2.44 3.38

Table 4. Average number of seeds / siliqua in OP and BP upon NaCl spray (3% and 5%) along with control (no spray) during different flowering regimes in the S-allele line II-12-4-7 (2010-2011). Conc.

3%

5%

Flowering Regimes

15 ( b ) OP BP

15 ( a ) OP BP

15 ( b + a ) OP BP

NS ( C ) OP BP

15 ( b ) OP BP

15 ( a ) OP BP

15 ( b + a ) OP BP

NS ( C ) OP BP

25-50% 50-75% >75%

1.11 0.32 0.48

0.46 1.33 1.33

0.66 1.35 1.56

0.00 0.00 0.00

1.10 0.54 0.62

0.54 2.42 2.65

2.60 1.24 2.09

0.00 0.00 0.00

3.86 0.75 3.71

3.28 1.57 3.35

2.95 2.52 0.72

3.23 2.43 1.81

2.43 2.74 1.87

OP – Self pollination in open flower stage, BP – Self pollination in bud stage, NS (C) – No water spray (control) 15 (b) - Spray of NaCl 15 minutes before pollination 15 (a) - Spray of NaCl 15 minutes after pollination, 15 (b+a) - Spray of NaCl 15 minutes before + 15 minutes after pollination

4.76 2.37 5.02

3.37 2.91 3.33

4.37 3.20 2.43

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Table 5. Number of seeds/siliqua as per completely randomized block design (CRBD) recorded in OP treatments in the S-allele lines I-4-6 and II 12-4-7. Treatments Concentration 3% 5% CD ( at 5% level ) Flowering regimes 25-50% 50-75% >75% CD (at 5 % level ) Time of sprays 15 ( b ) 15( a ) 15( b+a ) CD ( at 5% level )

Seeds/Siliqua I-4-6 3.58 3.44 NS

Seeds/Siliqua II-12-4-7 0.94 1.48 0.442

3.22 4.43 2.88 NS

1.02 1.18 1.44 NS

2.78 3.92 3.83 NS

0.68 1.42 1.53 0.54

Table 6: Number of seeds/siliqua as per CRBD recorded in OP treatments in the interaction (flowering regime x time of NaCl spray) in the S-allele line II12-4-7 Time of spray Flowering regime 25-50% 50-75% >75%

15 ( b )

15 ( a )

15 ( b+a )

1.10 0.43 0.53

0.48 1.82 1.95

1.46 1.30 1.85

CD: 0.9364 OP- Self pollination in open flower stage, CD- Critical difference 15 (b) - Spray of NaCl 15 minutes before pollination 15 (a) - Spray of NaCl 15 minutes after pollination 15 (b+a) - Spray of NaCl 15 minutes before + 15 minutes after pollination

findings are inconsistent with those of Fu et al. (1992) who had reported higher compatibility index than the control (no spray) in the self-incompatible plants of Brassica napus even up to 120 hours of common salt spray. The findings are also inconsistent with Kucera (1990) and Kucera and Cerny (1991) who had succeeded in maintaining the selfincompatible plants/lines of

DISCUSSION Erratic seed-set recorded during 2009-2010 in OP treatments in the S-allele line I-4-6 after the common salt sprays (3% and 5%) may be attributed to too long time lag between spray and pollinations and also to relatively lesser quantity (25 ml/plant) of NaCl solution sprayed on t the plants of the S-allele line. The present

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cauliflower and kohlrabi respectively, with sodium chloride (3%) solution sprays when pollinations were carried out 0.51.0 hour before or after NaCl spray. The findings are also inconsistent with Koprna et al. (2005) and Kucera et al. (2006) who verified the seed production of S-allele lines by spraying flowers with 5% and 3% NaCl solution respectively. The reason of getting practically no seed-set during 2009-2010 in OP treatments on the S-allele line II-12-4-7 despite common salt sprays could be the presence of stronger S-allele in this S-allele line as compared to the Sallele line I-4-6. During 2010-2011, good amount of seed-set was obtained in OP treatment on all the test plants, across the flowering regimes (2550%, 50-75% and >75% flowering), after the common salt (NaCl) sprays (3% and 5%) made 15 minutes before pollination, 15 minutes after pollination and 15 minutes before plus after pollination in the S-allele lines I-46 and II-12-4-7. The present findings (2010-2011), in which variable seed-set in OP (selfing in open flower stage) carried out before/after /before plus after common salt sprays (3% and 5%) were recorded, are in accordance with Kucera (1990), Kucera and Cerny (1991), Liao (1995) and Chaozhi et al. (2009) who had worked on temporary breakdown of self-incompatibility with common salt sprays in cauliflower, kohlrabi, cabbage and B. napus respectively. Kucera (1990) recorded 4.3 seeds per pod on the self-incompatible plants with common salt spray of 3% made

0.5-1.0 hour after self pollination as compared to 0.4 seeds per pod in the untreated plants. Kucera and Cerny (1991) found 3% concentration of common salt spray better than 1.5%. Spray before pollination was effective in one of the lines only. Liao (1995) obtained variable number of seeds in self pollination in different Sallele lines of cabbage sprayed with 3 per cent and 5 per cent common salt and pollinations made 10-15 minutes before and after spray. In one of the S-allele lines (No. 86193-5-3), 5% NaCl sprays proved better. In two of the S-allele lines i.e. No.15 and No.220, there were a very few seeds even after NaCl sprays probably due to the presence of stronger S-alleles in these lines. Chaozhi et al. (2009) recorded 7.03 seeds/siliqua after 35 % spray of NaCl on the S-allele line S-1300 of B. napus followed by hand pollination. Similarly, Wang et al. (2012) also reported the use of NaCl for overcoming self-incompatibility in non heading Chinese cabbage. Analysis of variance for the seed-set in OP during 20102011 in the S-allele line I-4-6 revealed that no source of variation (concentration of common salt, flowering regimes and time of common salt spray) was significant (Table 5). The coefficient of variation (CV) was rather very high (72.55%) which may be due to wide plant to plant variations in seed-set on the test plants. It is quite explainable since the present pollinations were carried out in insect proof nylon net enclosures under field conditions with no control over temperature and humidity/rains. Environmental

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common salt spray were at par in the S-allele line I-4-6 whereas 5% concentration was significantly superior in the S-allele line II-12-47 for seed-set in OP. The NaCl sprays made 15 minutes after and 15 minutes before plus after pollination were significantly superior to common salt sprays made 15 minutes before pollination in the S-allele line II-12-4-7. In general, seed-set in BP on the plants receiving common salt sprays were higher than the control plants (no spray).

factors especially temperature (Gonai and Hinata, 1971) and humidity (Carter and McNeilly, 1975) have been reported to influence the level of selfincompatibility in Brassica oleracea. Analysis of variance for the seed-set in OP in the S-allele line II-12-4-7 revealed that the common salt concentration of 5% was significantly superior to 3% concentration which may be attributed to the presence of stronger S-allele in the S-allele line II-12-4-7. The common salt sprays made 15 minutes after pollination and 15 minute before plus after pollination were at par with each other but significantly better than common salt sprays made 15 minutes before pollination (Table 5). Liao (1995) has reported that the number of seeds/siliqua depend on the kind of inbred line and degree of incompatibility, which might be the reason for getting relatively less number of seeds/siliqua in the S-allele line II12-4-7 as compared to the S-allele line I-4-6. The interaction between flowering regimes and time of common spray was significant in the S-allele line II-12-4-7 (Table 6). The interactions of the flowering regime >75% with common salt sprays made 15 minutes after pollination and 15 minutes before plus after pollination were at par but significantly superior to common salt sprays made 15 minutes before pollination. It can be concluded that common salt sprays (3% and 5%) proved effective in temporary breakdown of self-incompatibility in the S-allele lines I-4-6 and II-124-7. Both the concentrations of

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