Studies On The Selection Of Indeterminate Growth Habit Tomato Mutant Induced By Space Flight And Its Mutagenesis Mechanism

Space mutation breeding is a newly emerged breeding method, which consists of taking the seeds or other plant parts into the space by the recoverable satellite, where synthetically physical factors of the space are applied to create great genetic variations in them. Resultantly, many new kinds of germplasms were obtained, which were impossible through any conventional breeding method on the earth. There are several merits in space mutation breeding, such as its wider scope in variation, outstanding mutants obtained, higher frequency of mutation, and shorter period of the breeding program etc. Up to now, more than 70 crops and 500 cultivars had been sent to space for scientific research. As a result, more than 50 new cultivars or strains have been gradually derived via space mutation in China till now. In our present studies, we found a tomato plant with the indeterminate growth habit other than the determinate growth habit in its background parent (CK), through the space inducing mutation via the recoverable satellite, Shenzhou 4, which was sent to space in 2002. Continuous self-crossing and selection for three generations, a mutant with the stable indeterminate growth habit, MSI, was developed. In this paper, the process for the breeding of MSI, the morphological and physiological characters of the mutant MSI, as well as its cytological and molecular characters were studied, thorough the comparative analysis between MSI and its background parent, in order to open out a comprehensive look about the space mutation mechanism. The main results were as follows:1. Bred for of an indeterminate growth habit tomato mutant via space mutation In SP₁ population coming back from space, a special plant, SP₁-1, was found having higher plant height than others plants, being indeterminate growth habit other than the determinate one of its background CK. When CK was self-prunning (the average height was 103 cm), SP₁-1 plant was 145 cm, 42 cm higher than that of CK. All the plants in SP₂ population from the self-crossed seeds of SP|-1 were indeterminate growth habit. After continuous self-crossing and selection for three generation, a stable indeterminate growth tomato germplasm wasobtained.2. Morphologic characters and economic characters of MSIAt the time of self-prunning in CK population, the average plant height in MSI was 138cm, which was 33.40% higher than that of CK. The difference was obvious in the position of the 1st fruit node, which was 8.1 in MSI and was 1.5 higher than that of CK. The main stem diameter of MSI was 1.22 cm, 0.88 cm more than that of CK. Being the indeterminate growth habit and not self-prunning, the yield in MS 1 was much higher than that of CK, if the growth condition was good enough. The early yield and total yield of MSI were 43653.0 kg-hm"2 and 71253.8 kg-hm"2 respectively, which were 7716.8 kg-hm"2 and 18451.5 kg-hm'2 more than that of CK, respectively. The differences of the characters described above were obvious. Other characters such as fruit shape, signal fruit weight and fruit color in MSI were almost the same with CK.3. Observation of cytological characters in SPi-1 and MSIUsing the second root tips which were produced during the tissue culture of the indeterminate growth habit mutant and the root tips of MS 1, the chromosome configuration in mitosis and chromosome behavior in meiosis was observed. As the result showed, the chromosome number of the mutation plant SPpl was 24, the sameness as that of CK. The variation of the chromosome number and structure in SP|-1 and MSI has not been found in this experiment. The meiosis of pollen mother cell (PMC) in SP|-1 and MSI were also observed in this experiment. As a result, the 1st and 2nd cell division of meiosis in SPj-1 and MSI were both normal. No any chromosome segments or bridges and lagging chromosomes were found in the PMC meiosis. The size and structure of the pollens in SPj-1 and MSI was normal and no misshape pollens were observed as well.4. Enzymogram and activity of isoenzyme in MSIThe results showed that, the SOD enzymogram of MSI had not any difference comparing to CK. Both of them had three bands, and the positions of the bands were same as well. Although the number of POD bands between MSI and CK was the same, the staining extent had some distinct differences. One of POD isoenzyme bands in CK, Rf=0.512, was stained much darker than that of MS 1. The results of SOD and POD activities revealed that, there was no distinct difference of SOD activity between MSI and CK, which in MSI and CK were 24.83 u-g'1 and 24.52 u ? g"11 respectively. However, POD activities between MS 1 and CK were quite different,which in MSI and CK were 7.41 u-g"1 -min'1 and 9.76 u-g"1 -min"1 respectively, i.e. POD activity in MS 1 was much lower than that of CK.5. Photosynthesis characters in MSIThe results showed that, the tendencies of net photosynthetic rates (Pn) daily in MSI and CK were almost the same, with double peaks in the curves. However, the net daily photosynthetic rate (Pn) of MS 1 was much higher than that of CK especially in the period of photosynthetic fastigium. Similarly, the daily variation of stomata conductance (Gs) in MS 1 and CK had significantly differences and the curves were high at noon and low in the morning and afternoon. The average Gs in MSI was 0.254 mol-m" -s"1, which was much higher than that of CK. For the intracellular CO2 (Ci), their daily variation curves were shapes like letter "U". Contrast to Gs, Ci in MSI and CK had no distinct difference. The results of chlorophyll content analysis illustrated that the total chlorophyll contents in MSI was higher than that of CK, especially the chlorophyll-a, but the content of chlorophyll-b in MSI was almost the same to CK. This led to the increase of the ratio of chlorophyll-a/chlorophyll-b in MSI remarkably.6. Molecular marker analysis for MSIThe results showed that, 44 of total 50 10-mer Sangon RAPD primers amplified products and 2 primers of them, SI 65 and SI 68, amplified polymorphic products. The molecular weight of the specific amplified products were 300bp and 1500bp respectively, therefore they were named as TRSI65300 and TRS1681500 temporarily. Both of them were sequenced and transformed to SCAR markers, only of them, TRS 1681500, was a stable SCAR marker that could be used in genetic marker of the indeterminate growth habit mutant and MAS.7. The plants regeneration ability of MSIThe mutation plant obtained by space mutation was preserved by the method of tissue culture. During tissue culture, it was found that, when 6-BA concentration was 2.0 mg-L'1 in the media, the calli of the mutation explants was induced in 16 days and the callus inducing rate was 90%. However, at the same condition of tissue culture, the callus inducing rate was only 50% in 16 days of the culture. When IAA concentration in the media was increased gradually, the ability of callus inducing rate between the mutation and CK explants was same gradually, so did as the rate of plant regeneration. It was probable that, via space flight, there were some variation in the hormone concentration in the plant of MS 1, which led to the difference in the rate of callusinducing and plants regeneration between MS 1 and CK. 8. Studies on the hormone contents in MSI plantThe contents of 4 kinds of hormones, IAA, GA3, ABA and ZR, in the MSI plants during the plant growth season were assayed by ELISA. The results showed as follows: (1) In vivo IAA concentration of both MS 1 and CK increased gradually in early period of the plant growth, but in late season, that in MS 1 was increasing persistently, much different to that of CK in which the IAA content was tended to be a stable level. In the whole growth season, there was a little difference of IAA content between MSI and CK. (2) In early season, GA3 content in both MSI and CK was low. With the plants growing, GA3 content in MS 1 increased greatly, which was much higher than that of CK. (3) For ABA content, there was no obvious difference between MSI and CK during the early stage of plant growing. ABA content in the two kinds of plant was about 100-200 ng-mL"1. However, with the plants growing, ABA content in CK was increasing continuously. So ABA content in CK was much higher than that of MSI in the end. (4) In all the assayed period, ZR content in MS 1 was much higher than that of CK. And the difference was very significant. The results showed that the hormone content in MSI was varied greatly after treated by space flight. It was concluded that the expression of some genes related to the plant growing was changed which led to the variation in plant traits, although we had not found very powerful evidences to illuminate the reasons or causes of the mutation.