Study on dormancy and germination of thornless bone seeds

Study on dormancy and germination of thornless bone seeds

Fructus Lycii is a natural variety of Holly in Holly family, evergreen shrubs or small trees, tree species luxuriant, leaves strange, leaves dark green and glossy. The crown is round, yellow-green flowers bloom from April to May, inflorescences are Cymes or umbels, dioecious, drupe globose, initially green, mature in autumn, full of fruits, bright and dazzling, not withered by winter to the next spring, after pruning and shaping can be made into a large tree-shaped, spherical and tree-shaped bonsai, is a good ornamental, fruit, leaf and shape of the tree species, mainly used in the garden and urban greening.

As the dormancy cycle of the seeds of Fructus Lycii is generally as long as 3 years, it greatly affects the reproductive efficiency in production. How to break the seed dormancy quickly and improve the seed germination rate is the difficulty in the research of sowing and propagation of Fructus Lycii. In this paper, through the in-depth understanding of thornless bone seeds, to explore the factors affecting seed dormancy, to find a reasonable way to quickly break seed dormancy, and to lay a foundation for other holly species how to break dormancy.

1 materials and methods

1.1 Materials

The thornless bone seeds for use are the fresh seeds of the year purchased from the market in November 2016. the drupe was soaked in water for 2 days. After the pulp was fully soft and ripe, the seeds were scrubbed, washed, the floating seeds in the upper layer were removed, and the full seeds sunk in the lower layer were used as experimental materials.

1.2 method

The main results are as follows: (1) determination of 1000-seed weight, morphology and vitality. Determination of 1000-seed weight and morphology of seeds: the 100-seed method was used to determine the 1000-seed weight and morphology of seeds. 100 seeds were used as a group with five repeats. The length and width of seed, the weight of seed shell and the size of endosperm were detected and recorded.

Determination of seed viability: 2 × 100 selected seeds were randomly selected, soaked in water for 48 hours, and the top of the seed was cut off horizontally. Then the seed was placed in a 35 ℃ lightless incubator and stained with 0.2% tetrazolium solution for 12 hours. After dyeing, the seed coat was washed repeatedly, the seed coat was stripped, the seed was cut longitudinally along the central axis, and the embryo was exposed. The identification criteria are:

① viable seeds: all embryos and endosperms were stained normally, all embryos were stained, and a few endosperms were not stained.

② seeds with no viability: the embryo and endosperm were not stained, but all the embryos were stained and the endosperm was not stained.

(2) determination of water absorption of seeds. Two air-dried seeds were taken, 60 seeds were taken as one, the first was intact air-dried seeds, and the second was punctured with a needle. The two seeds were placed in a 25 ℃ constant temperature incubator to absorb water, and the weights were measured at 2, 4, 6, 8, 10 and 12 h respectively. When taking out the seed, the absorbent paper thoroughly absorbs the floating water on the seed surface, then place the 10min to weigh and record it.

(3) concentrated sulfuric acid treatment. The seeds for the test were poured into a certain amount of concentrated sulfuric acid (specific gravity 1.84) solution, and then treated for different time (1 min, 5 min, 10 min, 10 min, 30 min, 1 h, 2 h, 3 h), and then washed in running water for 16 hours, and then germinated. The treatment was repeated three times with 20 seeds in a group.

(4) Chemical treatment. The holly seeds were treated with GA3 solution, soaked in different concentrations of GA3+H2SO4 solution (100250500mg/L) for 24 hours, and then germinated. The treatment was repeated three times with 20 seeds in a group.

(5) stratification treatment. Natural level: on January 1st, seeds and wet sand were layered and stacked in flowerpots (the ratio of wet sand to seeds was 3 ∶ 1). The mouth of the pot was fastened with plastic sheeting and placed outside in a dry and cool place with good drainage and deep 20cm to accelerate germination for about 180 days. on July 1st, the seeds were taken out to calculate the germination rate.

Low temperature stage: on January 1, the seeds and wet sand were stacked in layers (the ratio of wet sand to seeds was 3 ∶ 1) in the flowerpot, the mouth of the pot was fastened with plastic sheet, placed in the indoor refrigerator for 180 days, the temperature was 4 ℃, and the germination rate was counted on July 1. The treatment was repeated three times.

Stratification of seeds and wet sand (the ratio of wet sand to seeds is 3 ∶ 1) was stacked in a flowerpot on January 1. The mouth of the pot was tied up with plastic sheeting and placed in the indoor refrigerator for 120 days at a temperature of 4 ℃. It was taken out of the refrigerator on May 1st and placed in a dry, cool place with good drainage and deep 20cm for 60 days. The germination rate was counted. Repeat three times.

1.3 Statistical analysis of data

The data were analyzed by Excel and SAS software. Germination rate (%) = number of germinated seeds / number of tested seeds × 100% water absorption (%) = [quality after water absorption (g)-quality before water absorption (g)] / mass before water absorption (g) × 100%

2 results and analysis

2.1 Biological characteristics of thornless bone seeds

The main results are as follows: (1) the basic morphological characteristics of thornless bone seeds. The thornless bone fruit is berrylike, usually spherical, and the ripe red fruit hangs from the branches and turns crimson when dried. The pulp is thick and succulent. Each fruit has about 4-5 seeds, the seeds are narrow and lanceolate, and the outer back is shaped like a gully. The clean and good seeds were obtained by kneading and cleaning the thornless bone fruit. through the determination and observation of the length, width and endosperm of the seeds, the following data were obtained, as shown in figure 1.

Fig. 1 seed morphology and embryo development of thornless bone

Ⅰ and Ⅱ are the morphological characteristics of the outer skin of the unthorn bone seed, and Ⅲ is the endosperm of the unthorn bone seed.

The external shape of the seed is uniform, the length is (6.64 ±0.35) mm, the width is (3.86 ±0.32) mm, the height is (3.72 ±0.25) mm, the 1000-seed weight is 15.22g, the seed kernel rate is 78.26%, and the seed shell weight accounts for 81% of the complete thornless bone seed. The results show that the thickness of the seed shell leads to seed dormancy to some extent.

(2) determination of seed viability. Seed vitality is not only the basic standard for testing the growth and development of seeds, but also reflects the quality of seeds. After testing, there are 187 seeds with vitality and 13 seeds without vitality. The viable seeds accounted for 93.5% of the total number of seeds, indicating that most of the seeds tested were of good quality.

(3) the water permeability of seeds. The water absorption of intact seeds and broken seeds is shown in figure 2.

Fig. 2 Water absorption curve of thornless bone seeds

As shown in figure 2, in the early stage of soaking, the water absorption of intact seeds was basically the same as that of broken seeds. With the passage of time, the water absorption of barbed seeds without thorns was higher than that of intact seeds in the middle stage of soaking, and in the later stage of soaking, the water absorption of the two kinds of seeds reached the same saturation. The results showed that because the seed coat of thornless bone seed was hard, the water absorption of intact seeds increased slowly, and the hardness of seed coat affected the water absorption of seeds to a certain extent.

2.2 effects of different acid etching time treatments on seed germination percentage due to the hardening of the seed coat of thornless bone seeds, which hindered the seed germination, but the acid etching treatment could reduce the seed shell, increase its permeability and improve the seed germination rate. Through the acid etching treatment of thornless bone seeds at different times, the most suitable acid etching time can be found, as shown in Table 1 below.

According to Table 1, after acid etching, the longer the acid etching time is, the more the seed shell corrosion ratio is, and the decay rate is relatively increased, while the germination rate changes with the change of seed shell corrosion ratio and decay rate. The results showed that after acid etching for 1 hour, the unthorn bone seeds could more appropriately reduce the weight of seed shell, stabilize the degree of seed decay and promote the germination of unthorn bone seeds.

Table 1 effect of different acid etching time on germination rate of thornless bone seeds

2.3 effect of comprehensive treatment of GA3 and concentrated sulfuric acid on seed germination rate

Table 2 effects of different concentrations of GA3 on the germination rate of thornless bone seeds

According to Table 2, the comprehensive treatment of different concentrations of GA3 solution and different acid etching time significantly increased the germination rate of unthorn bone seeds, and the germination rate increased with the increase of GA3 concentration, but it was not that the higher the concentration, the more favorable, but too high concentration decreased the activity of non-thorn bone seed embryos. The results showed that the germination effect of seeds treated with 250ml GA3 concentration was the best.

2.4 effect of stratification treatment on seed germination rate

Stratification treatment is an effective method to break the comprehensive dormancy of seeds. Stratification treatment can be divided into natural stratification, low temperature stratification and variable temperature stratification. Through different stratification treatments (natural stratification, low temperature stratification 180 days, variable temperature stratification warm temperature 60 days + low temperature 120 days), the effect on the germination rate of thornless bone seeds was discussed, as shown in figure 3.

Fig. 3 effects of different stratification treatments on seed germination rate

The experimental results show that after 180 days of natural stratification and low temperature stratification, the germination rate of thornless bone seeds is 0.6% and 1.3% respectively. Compared with natural stratification and low temperature stratification, the germination rate of variable temperature stratification is obviously superior, and the germination rate is up to 5.8%. Therefore, it can be inferred that thornless bone seeds have no obvious promoting effect on breaking dormancy after natural stratification, compared with natural stratification. Low temperature stratification can break the dormancy of some seeds, but the germination rate is also low. however, the thornless bone seeds treated by variable temperature stratification (warm temperature first and then low temperature) are more beneficial to the growth and development of seed embryos and improve the germination rate of seeds.

3 discussion and conclusion

3.1 discussion

(1) the effect of seed coat on seed dormancy. A series of seed coat obstacles, such as Lignification, hardness and compactness, lead to forced dormancy of seeds. Generally speaking, seed coat disturbance usually refers to the impermeability and air permeability of seed coat and mechanical obstacles caused by seed coat. The hard seed coat limits the gas exchange between the seed embryo and the outside world, blocks the emission of oxygen and carbon dioxide, hinders the breathing of the seed embryo, and is unable to provide the necessary energy for seed germination. the seed coat characteristics of hard Lignification and dense cell arrangement are the main factors leading to seed dormancy, according to Li Jinyu's study on walnut seeds. The mechanical restraint of seed shell is the main reason for slow seed germination. The poor air permeability of the hard and dense seed coat of Ilex fargesii is one of the factors of forced dormancy of seeds. Xu Benmei et al pointed out that the forced dormancy of big fruit seeds is mainly caused by poor air permeability of seed coat. This characteristic is analyzed and compared with the similar theories of Hawthorn (Crata-egus pinnatifida bunge.), round cypress (Sabina chinensis), rose (Rosa laevigata), weight hammer (S.xylocarpa), big leaf holly, bladder fruit (S.holocarpa) and so on. In this experiment, the water permeability, air permeability and mechanical obstacles of the seed coat hindered the seed germination to a great extent and inhibited the growth of the embryo.

Acid etching treatment can effectively etch the dense tissue cells outside the seed coat, reduce the seed coat obstacle and the weight of the seed coat, enable the embryo to break away from the bondage of the seed coat, enhance the embryo permeability, and promote the germination of the seed embryo. After the thornless bone seeds were treated with concentrated sulfuric acid, the seed germination rate was greatly improved, and it is very important to grasp the appropriate acid etching time, if the acid etching time is not enough, the seed coat barrier can not be fully etched. In order to achieve the expected effect of seed germination, if the acid etching time is too long, the seed coat will be fully eroded, causing irreparable damage to the seed embryo, which is easy to rot and lead to the death of the seed embryo.

(2) the effect of embryo development on seed dormancy. After the thornless bone fruit is ripe, the fruit is picked, the fruit is rubbed and the seed is dried in order to dissect the seed. As shown in figure 1, the length of the seed embryo is about 0.3~0.4mm, which is only 1 / 10 of that of the embryo at the time of germination, and the embryo is still in the embryonic primordium stage. The immature embryo absorbs energy from the nutrients of the endosperm to promote seed germination. after the seed is matured naturally, the seed embryo is still in the primary stage and the embryo is not fully developed. at this time, it is necessary to complete morphological development and maturity in order to break dormancy and promote germination.

For how to speed up the physiological post-ripening process of thornless bone seed embryo, variable temperature stratification treatment is the most effective method. In the warm stratification stage, the seed embryo begins to differentiate slowly, and the seed embryo grows and develops slowly, which is consistent with the conclusion obtained by Leonard et al in the study of American ginseng seed. In the process of warm stratification, the embryo may have a static period for a period of time, so the endosperm needs to absorb a lot of nutrients from the seed embryo in the early stage of warm stratification, so the seed embryo grows slowly. In the stage of low temperature stratification, the seed embryos began to grow rapidly, and after 120 days of stratification, most of the thornless bone seeds had completed the process of physiological post-ripening, but the difference was that the quality and development of various embryos were different. a small number of unthorny bone seeds are still in a semi-mature state, resulting in inconsistent germination homogeneity of unthorn bone seeds.

(3) the effect of endogenous inhibitors on seed dormancy. There are germination inhibitors in the fruit and seed coat of many plants, mainly including some organic acids, alkaloids, lipids, aldehydes and so on, which can directly or indirectly inhibit seed germination. Coumarin and abscisic acid are two strong inhibitors to seed germination.

GA3 treatment can effectively promote the growth and development of seed embryos. It can induce endosperm to produce hydrolytic enzymes and convert storage substances in water from macromolecules to small molecules, such as starch hydrolysis to sugar and protein hydrolysis to amino acids for embryo absorption. At the same time, it can promote the conversion between organic and inorganic substances in the embryo, make the embryo complete the physiological post-ripening process, and promote cell division and elongation. Promoting the synthesis of DNA and RNA starch [Jishan Huayao] can inhibit the accumulation of ABA in dormant seeds.

3.2 conclusion

Through a series of discussions and studies on thorn-free bone seeds, to find out the main factors affecting seed dormancy, thornless bone seeds have the characteristics of hard seed coat, seed shell accounts for 87.3% of seed weight, and air permeability is extremely poor. affecting the material and gas exchange between seed embryo and endosperm and hindering seed germination is one of the main factors leading to seed dormancy. Acid etching treatment can etch the seed coat, break the seed coat barrier and accelerate the growth and development of the seed embryo, but it can not make the seed germinate ahead of time. There are some endogenous inhibitors in the embryo and endosperm of the seed, which limit the growth and development of the embryo to a certain extent. Gibberellin GA3 can reduce the endogenous inhibitor in the embryo and promote the growth and development of the embryo. In addition, because the thornless bone seed has the characteristic of germination every other year. After picking, the fruit is not fully developed and can only germinate after physiological ripening. Stratification is a commonly used method to break seed dormancy, which is divided into natural stratification, low temperature stratification and variable temperature stratification. Thornless bone seeds have physiological characteristics of germination every other year and seed coat characteristics of physiological post-ripening, and natural stratification can not break seed dormancy in a short time. Low temperature stratification can only break the dormancy of some mature seeds, and the germination rate is low. Variable temperature stratification (warm temperature first and then low temperature) treatment has a very significant change in breaking seed dormancy, which can accelerate seed growth and development, accelerate seed embryo absorption and promote seed germination. it is the most suitable stratification treatment to break seed dormancy. Therefore, after understanding the various factors that hinder the dormancy of unarmed bone seeds, the best method to relieve seed dormancy is as follows: soaking seeds in 98% concentrated 1h+250mg/L gibberellin solution for 24 hours + warm stratification for 60 days + low temperature stratification for 120 days.