IDRC2ตอนที่2
The Uses of Self-incompatibity genes on Chinese Cabbage
Nikornpun, M.1 and E. Payakhapaab1
ABSTRACT
Results showed that Chinese cabbage line 40-9 gave the highest seed weight of 0.165 gram per plant when self-pollinated. Seed set analysis and fluorescent microscope techniques were used to check self-incompatibility of 9 Chinese cabbage lines. Completely randomized design was used. Self-incompatible lines were detected on 23-3-4, 27,27-3-7 and 40-9 lines when tested by using seed set analysis. However, when fluorescent microscope technique was used, not all lines were self-incompatible, lines 23, 23-3-1, 40 and 142-5 were also found to be weak self-incompatible. Four inbred lines with self-incompatibility were selected to produce seed. Reciprocal crosses (by bee pollination) showed that when line 40-9 was used as a female parent, they gave the highest seed yield which was a maternal effect. Crosses of 40-9 x 142-5, 40-9 x 27-3-7 and 40-9 x 23-3-4 gave high seed weight : 4.8, 3.9 and 2.7 kg/rai, respectively. Eleven hybrid varieties of Chinese cabbage were tested in comparison with 3 commercial varieties in winter 1998 at Chiang Mai University. Hybrid of 142-5 x 40-9 gave the highest head yield of 6,170 kg/rai (6.25 rai = 1 ha) with 36.3% higher yield than the commercial varieties. Other crosses such as hybrids 23 x 27, 23 x 142, 27-3-7 x 23-3-4, 27-3-7 x 142-5 and 40-9 x 23-3-4 had good horticultural characteristics. They yielded 4,646, 5,551, 3,665, 5,150 and 3,785 kg/rai, respectively.
Keyword : self-incompatibility, seed set analysis and fluorescent microscope technique.
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1Faculty of Agriculture, Chiang Mai University. Chiang Mai, Thailand, 50200.
INTRODUCTION
Chinese cabbage is not only a popular vegetable but also an economical crop for export. Chinese cabbage seeds used in Thailand are both open pollinated and F1 hybrid varieties and are all imported. Popular Chinese cabbage varieties which farmer grow are hybrid varieties from Japan, Taiwan and Korea.
Self-incompatibility is a special characteristic of some Brassica crops which promotes cross-pollination and prevents self-pollination of these crops. This characteristic is controlled by multiple allelic genes at a single locus. Pollen tube germination was inhibited by stigma if they carried same self-incompatible genes (Nasrallah and Nasrallah, 1993, Nasrallah et al, 1994 and Isogai et al, 1987). The reaction is a sporophytic reaction which 2n chromosome of S locus of both parents control the reaction (Gaude et al, 1993, Pastuglia et al, 1997 and Nasrallah and Nasrallah, 1993). Chinese cabbage also carries self-incompatible genes of S locus. It is classified into a sporophytic reaction (Opena et al, 1988). The self-incompatible genes will be used to develop F1 hybrid Chinese cabbage for high yield and good quality. Additionally, bee pollination is also studied, for F1 hybrid seed production. The pollination is required to replace labor used in parental lines and F1 hybrid seed production.
MATERIALS AND METHODS
1. Hand pollination of unopened flower bud for inbred seed production of Chinese cabbage.
Inbred lines of Chinese cabbage No.23-3-4, 27-3-7, 40-9 and 142-5 were germinated for two days, then they were vernalized at 4-5°C for 15 days before planting in the field. Inflorescence was covered with a paper bag before blooming. When some flowers of the inflorescence started to open, the paper bag was taken off. Young flowers (unopened) were forced to open using forceps. The pollen from opened flowers of the same inflorescence was used to pollinate the young flowers. All opened flowers were taken off. Inflorescence was covered with the same paper bag. Seeds were collected as they matured. These seeds are considered as inbred seeds.
2. Testing for self-incompatibility levels of Chinese cabbage.
Two methods to test self-incompatibility of Chinese cabbage were used; seed set analysis and fluorescent microscope technique. Seed set analysis method is a common method for testing self-incompatibility (Shinohara, 1981 and Opena et al. 1988). Three to four inflorescences per plant were bagged before flower opened. Three to five days later, the bags were taken off. Unopened and opened flowers were marked with a thread. Emasculation was needed for all flowers tested. They were pollinated with pollen of the same plant. After pollination, the inflorescences were bagged for two weeks. About 10 plants were used for an inbred line, then the pods were counted, the young flowers should be able to bear seeds while the opened flowers should not be able to bear seeds if they are self-incompatible.
Fluorescent microscope technique was used to test self-incompatibility levels ( Kho and Baer, 1968 ). Unopened and opened flowers were taken from the same plant. They were put on a slide in a petridish containing potassium dichromate solution. The potassium dichromate (K2Cr2O7 ) when boiled and kept in a tight chamber, will bring about the level of humidity to 98 %RH. The flowers were emasculated before pollination with pollen from the same plants. Another opened flower was required on the slide, it was cross-pollinated with pollen from different varieties, it was used as a control flower. The flowers were kept in the petridish for one night. Styles of these flowers were sectioned in sodium hydroxide (NaOH) which was heated to 60oC for one hour. Then the styles were stained with 0.2 % aniline blue in 2% potassium phosphate (K3PO4.3H2O) for 24 hours. They were squashed on a slide which had a drop of glycerol. Pollen tubes were counted using a fluorescent microscope.
3. F1 hybrid seed production by bees of Chinese cabbage.
Four inbred lines of Chinese cabbage, 23-3-4, 27-3-7, 40-9 and 142-5 were vernalized at 4-5°C for 15 days. They were planted in a field at 30 cm between plants and 50 cm between rows. Six seedlings were planted for each line, 3 plants per row. A plot size of 2 x 2 m was used, two plots per crossing. Each plot was planted with 2 inbred lines, side by side. A salan net was used for covering the plots from insects and keeping honey bees inside the net. Twelve crosses were set up, 23-3-4 x 27-3-7, 23-3-4 x 40-9, 23-3-4 x 142-5, 27-3-7 x 23-3-4, 27-3-7 x 40-9, 27-3-7 x 142-5, 40-9 x 23-3-4, 40-9 x 27-3-7, 40-9 x 142-5, 142-5 x 23-3-4,142-5 x 27-3-7 and 142-5 x 40-9. A beehive was put in each cross. Mature seeds were harvested when pods dried.
4. Evalution of F1 hybrid Chinese cabbage.
Randomized complete block design of 3 replications was used for this experiment. Eleven F1 hybrid varieties were tested against such control varieties as Chang, Bomb 159 and Tapa 23. The F1 hybrid varieties were 23 x 27, 23 x 142, 27 x 23, 27-3-7 x 23-3-4, 27-3-7 x 40-9, 27-3-7 x 142-5, 40-9 x 23-3-4, 40-9 x 27-3-7, 40-9 x 142-5, 142 x 23 and 142-5 x 40-9. Each treatment was planted in a 2 x 2 m plot. Eight plants were planted per plot at spacing of 40 x 50 cm. Guard rows were planted around each replication.
RESULTS
1. Hand pollination of unopened flower bud for inbred seed production of Chinese cabbage.
Inbred lines of Chinese cabbage were different in seed weight (Table 1). Line 40-9 gave the highest seed weight of 0.165 g/plant followed by lines 27-3-7 and 27 (0.153 and 0.123 g/plant), respectively. It was observed that unopened flowers in the middle of an inflorescence produced the highest seed numbers while unopened flowers at the top and bottom of the inflorescence produced a few seeds or did not set seed. It was observed that the flowers which gave good seed set were large in seed size but the yellow color of petals were not distinctly seen.
Table 1 Seed weight from self-pollinated Chinese cabbage.
Line / Seed weight/plant (g)40-9 / 0.165
27-3-7 / 0.153
27 / 0.123
23 / 0.076
142-5 / 0.047
23-3-4 / 0.032
2. Testing for self-incompatibility levels of Chinese cabbage.
2.1 Seed set analysis method.
Opened and unopened flowers of the same inflorescence were self-pollinated. When seeds were matured the numbers of seed set in the opened flowers indicated self-compatibility levels of the plant. Inbred lines 27-3-7 and 40-9 showed strong self-incompatibility. The range of 0-25% seed setting was used to indicate strong self-incompatibility levels.The strongest self-incompatible line was 27-3-7 which showed 22.01% seed set, followed by lines 40-9 and 23-3-4 having 24.72 and 29.24 % seed set, respectively. The range of weak self-incompatibility was set at 26-50% as indicated in lines 23, 27, 142 and 142-5 (Table 2 and Figure 1).
Table 2 Levels of self-incompatibility of Chinese cabbage lines, tested by seed set analysis.
/ Number of pod / Number of seedLine / Unopened / Opened / Unopened / Opened / Seed set1 / Conclusion2
flower / flower / flower / flower / (%)
23 / 15 / 15 / 210 / 80 / 38.10 / WSI
23-3-4 / 15 / 15 / 236 / 69 / 29.24 / WSI
27 / 15 / 15 / 193 / 96 / 49.74 / WSI
27-3-7 / 15 / 15 / 231 / 51 / 22.01 / SI
40-9 / 15 / 15 / 267 / 66 / 24.72 / SI
142 / 15 / 15 / 253 / 88 / 34.78 / WSI
142-5 / 15 / 15 / 245 / 79 / 32.24 / WSI
1 0-25% self-incompatibility, 26-50% weak self-incompatibility, 51-75% weak
self-compatibility and 76-100% self-compatibility.
2 SI = self-incompatibility
WSI = weak self-incompatibility