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Zhou Jialiang et al
Funds: General Project of the Key R&D Program of Jiangxi Provincial Department of Science and Technology "Research and Demonstration of Seedling Breeding Technology of Jinggang Pomelo Light and Simplified Virus-free Improved Seeds" (Project No.: 20202BBFL63012).
Citrus huanglongbing, one of the most important quarantine diseases in the world, originated in the Chaoshan region of Guangzhou and was first reported in southern China in the early 20th century. For a period of time, huanglongbing disease has broken out in areas with large citrus planting areas in mainland China, which has seriously affected the development of the citrus industry in mainland China. In the 60s of the 20th century, scholars at home and abroad had a heated discussion on the pathogen of huanglongbing, and at first, some scholars believed that the cause was virus infection or mycoplasma and other prokaryotes. It was not until the 20th century that scientists observed the pathogenic bacteria of huanglongbing under an electron microscope, and after that, the pathogenic nature of huanglongbing was gradually clarified. At present, one of the most effective methods to obtain detoxified seedlings is shoot tip microbud grafting, but in the actual production process, there are often unstable situations such as high grafting pollution rate and low germination rate, so it is meaningful to explore the best experimental conditions for this technology.
In this paper, we will study the optimal experimental conditions for microbud grafting experiments, i.e., to explore the best time for taking stem segments of pomelo in vitro to induce young shoots in vitro, and to explore the optimal graft size by using stem segments of different sizes in vitro.
1 Materials and methods
1.1 Experimental materials
1.1.1 Hitogi
The ripe fruits of Citrus aurantium husk harvested in autumn (October) were treated with 75% alcohol, the fruits were surface sterilized and packed in fresh-keeping bags, and cultivated into rootstock yellowing seedlings in test tubes.
1.1.2 Grafting materials
The stem material of Jinlan pomelo comes from the Jinggang pomelo breeding base in Jinggangshan Agricultural High-tech Industry Demonstration Zone, Ji'an City, Jiangxi Province. Stem segment disinfection was directly used for various treatments of microbud grafting test.
1.1.3 Stem tier
The age of each tested pomelo is less than 3 years, the materials are from the above-mentioned pomelo varieties, and the explants are the stem segments of the above adult pomelo, most of which are spring, summer and autumn shoots in the same year.
1.1.4 Culture medium
The plant growth regulator used in the experiment was 6-BA, and the other substances required in the medium were sucrose and agar. MT medium, MS medium required for a variety of trace or large elements.
1.2 Research Methods
1.2.1砧木黄化苗的培养
First, peel the full seeds from the ripe citrus aurantium fruits, soak the citrus aurantium seeds with 75% alcohol for 1min, and carefully rinse them with sterile water for 3 times, then carefully peel off the inner and outer seed coats of the seeds on the sterilized glass dish, disinfect the seeds with 5% sodium hypochlorite solution for 5~10min, and carefully rinse them with sterile water for 5 times; ensure that the treatment environment is sterile, and inoculate each tube of citrus aurantium seeds with solid medium MT+ sucrose.
Mark the date and treatment method, and place it in the dark culture room at 26 °C ± 2 °C for about 21 days in order to obtain yellowing seedlings that can be used for grafting, and put the yellowing seedlings in the culture room under light 1 to 2 days before grafting, and exercise them as grafted rootstocks. If it is not necessary to use it as a grafted rootstock in time, it can be stored in a 4°C refrigerator, and then put it in the culture room for temperature exercise 6 hours in advance when it needs to be used.
1.2.2 Stem segment disinfection test
The outer epidermis of the stem segment of Citrus maxima 'Jinlan Yu' contains exophytes that can be removed under appropriate disinfection treatment, but endophytes are difficult to kill by general disinfection methods. In order to solve these problems of adult materials in the early culture, the disinfection experiments were carried out on the stem segments by using different disinfection disinfectant combinations, disinfection time and disinfection combinations, in order to obtain better disinfection effects.
Table 1 Experimental design of stem disinfection in different seasons and different lengths
Table 2 Results of stem disinfection experiments in different seasons
Table 3 Results of disinfection experiments on stem segments of different sizes
The specific technology is as follows: select an excellent mother plant, cut the tender shoots containing 2~3 axillary buds into 5cm long segments, rinse with diluted dish soap solution first, and then rinse with running water for 30min to remove surface dust. Rinse several times with sterile water when operating on the ultra-clean bench and put it in a sterile beaker for later use. The culture was carried out at 26 °C±2 °C, with a photoperiod of 16 h/day and a light intensity of 2500 lx.
(1) Experimental design of the effect of disinfection of materials in different seasons.
At about 9 a.m. on a sunny day at the end of each month from March to October, the semi-lignified shoots of Citrus maxima 'Jinlan Yu' within 3 years were taken from Jinggang in Jinggang Honey Pomelo Breeding Base in Jinggangshan Agricultural High-tech Industry Demonstration Zone, sterilized with 5% NaClO for 10min, rinsed with sterile water 3 times, repeated once, and then inoculated in MT solid medium. The number of stem segments in each treatment was 30 stem segments, repeated 3 times, the survival rate of grafted seedlings after 10 days was observed and recorded, and the survival rate after 30 days was calculated.
(2) Experimental design of disinfection effect of different explant sizes.
Take the 5cm stem segment of Citrus maxima 'Jinlan Yu', soak it in 75% alcohol for 15 seconds on an ultra-clean workbench and shake it, sterilize it with 5% NaClO for 20min (divided into two times, 10min each time), keep shaking during the disinfection period, so that the disinfectant and the explant are fully combined, and rinse with sterile water 3 times each. The treated stem segments were cut into 1cm, 2cm, 3cm, 4cm, and 5cm respectively. Inoculated on MT+6-BA (1.0mg/L+40g/L) sucrose medium induced adventitious bud germination in stem segments. The number of stem segments in each treatment was 20 stem segments, repeated three times, the observation began after 5 days, the contamination rate was counted after 10 days, and the related growth was counted after 30 days.
2 Results and Analysis
2.1 Table 2 shows the results of stem disinfection effect in different seasons.
2.2 The results of disinfection experiments on stem segments of different lengths are shown in Table 3.
2.3 Comparison of disinfection pollution rate and germination rate of stem segments in different seasons
At the end of each month from March to October, around 9 a.m. on a sunny day, the stems grown within 3 years are disinfected. According to the experimental results of Fig. 2, the pollution rate obtained in late May was the lowest, about 23.33%, and the germination rate was the highest, about 73.33%, while the highest pollution rate was about 72.22% in late October, and the germination rate was the lowest, only 18.89%, and the lower pollution rate and higher germination rate in March~May, and the effect was worse in July~October (see Fig. 1).
Fig.1 Comparison of stem disinfection experiments in different seasons
Fig.2 Comparison of disinfection experiments with different stem segment sizes
2.4 Comparison of disinfection contamination rate and germination rate of stem segments of different sizes
According to Fig. 2, the pollution rate of 40.00% was the lowest when the stem length was 1~2cm, and the germination rate of the 2cm stem segment was the highest at 35.00%, and the pollution rate of the other stem segments was higher than that of the 1~2cm stem segment, and the germination rate was significantly lower than that of the 2cm stem segment.
According to the experimental data, the selection of stem segments in different seasons and different sizes as the materials for in vitro induction of sprouts has a significant effect on the contamination rate and germination rate of transplants, and the selection of explant disinfection methods also has a great impact on the disinfection effect.
The tender shoot segments in late May were sterilized with 5% NaClO for 10 min, rinsed with sterile water for 3 times, sterilized with 5% NaClO for 10 min, rinsed with sterile water for 3 times, and inoculated in MT solid medium. Among them, May is the best time to determine the timber harvesting period, with the lowest pollution rate of 23.33% and the highest germination rate of 71.11%. Analysis may be due to the low temperature in March, poor growth of spring shoots in early spring, although some of the scion survived, but the ability to divide and differentiate was weak, summer shoots in July, due to the increase in temperature, the concentration of bacteria in the air increased, resulting in the increase of bacteria on the surface of new shoots, and the difficulty of disinfection.
The stem segments of the explants were cut into 1cm, 2cm, 3cm, 4cm and 5cm respectively, and the results showed that the contamination rate of stem segments with a length of 1~2cm was the lowest, about 40.00%, and the germination rate of 2cm stem segments was the highest, about 35.00%. The reason for the analysis is that the size of the stem segment is directly proportional to the number of bacteria it carries, that is, the longer the stem segment, the greater the number of bacteria carried, and therefore, the worse the disinfection effect, and vice versa. The low germination rate of the 1cm stem segment may be due to the short stem segment and insufficient nutrient absorption.
3 Conclusion
According to the analysis and discussion of the experimental data, the results of ex vivo induction of explant stem segments in late May showed that the contamination rate was low, and the effect of using 2cm stem segments was the best.