Outdoor Domestication Cultivation and Survival Mechanism for Tissue Culture Seedlings of Paeonia suffruticosa

Xianglong MA1, Jingxu WU2*, Yixin LIANG1, Shaohua LIU3, Yongmei WANG

1. Luoyang Peony Development Center, Luoyang 470000, China; 2. Luoyang Fine Peony Garden (Mangshan Huafeng Horticulture Garden), Luoyang 471000, China; 3. Henan Forestry Vocational College, Luoyang 471002, China; 4. Luoyang Wangcheng Park, Luoyang 471000, China

Abstract In order to reveal the differences between different peony varieties and facilities, thousands of tissue culture seedlings of 21 Itoh peony varieties imported from abroad were used as test materials, and the peony domestication cultivation method used in the past was used as a control. The technical measures and mechanisms for the outdoor domestication of seedlings and the survival of field transplantation were studied. The results indicate that the main factors and measures for effectively protecting the outdoor transplanting of tissue culture seedlings include cultivating sound and strong seedlings, transplanting in proper period of spring, and providing good ventilating places and facilities, removing leaves, keeping buds and releasing dormancy before transplantation, planting depth should meet the requirement of exposing to the root neck, changing the pots according to the size of seedlings, the substrate should be loose and permeable, seriously disinfected and sterilized, scientific management, and prevention and control of diseases and insect pests. In addition, the survival mechanism was also analyzed, and the reasons and countermeasures for the successful application of peony tissue culture in China were found. Finally, the differences in survival rates between different varieties and facilities were summarized.

Key words Tissue culture seedlings of Paeonia suffruticosa, Outdoor domestication cultivation, Survival rate, Survival mechanism

1 Introduction

Paeoniasuffruticosais one of the most important flower species in China, and its low breeding rate and low speed are the key factors hindering its current industrialization. Tissue culture is internationally recognized as the fastest and most efficient propagation method. It is an important means of detoxification and breeding, solving the problem of germplasm degradation, and also a necessary foundation for transgenic engineering and molecular biology research. However, due to the specificity ofP.suffruticosaand other Paeonia plants, there are few reports about the application of tissue culture technology in the rapid propagation of seedlings. Many successful researches and reports are mostly limited to the laboratory stage. There are still no precedents for the successful application of multi-species large-scale tissue culture seedlings in field production. A core reason for this problem may be that the tissue culture seedlings are not successfully cultivated in outdoor cultivation and low survival rate of field transplantation.

In 2016-2017, the multi-variety and high-quality Itoh hybridP.suffruticosaprovided by Canada and traditionalP.suffruticosawere used as experimental materials, and the experimental research on outdoor cultivation and field cultivation of tissue culture seedlings was carried out, which was the first success in China. Through comparing with the domestically cultivatedP.suffruticosatissue culture seedlings in the past, we analyzed and summarized the survival mechanism ofP.suffruticosatissue culture seedlings, to reveal the differences between different varieties and facilities, in the hope of providing some recommendations and references for related works in China.

2 Materials and methods

2.1 MaterialsOn April 15, 2016, we introduced 1 500 plants of eightP.suffruticosavarieties, including Singing in the Rain, Kopper Kettle, Lollipop, and Red Doodle; on March 20, 2017, we introduced 3 600 plants of 21 varieties, including Smith Family Yellow, Pink Doodle, Red Doodle, Yellow Doodle, Black Swan, Singing in the Rain, Oochigeas, Impossible Dream, Magical Mystery Tour, Cora Louise, Lilac Purple, Duchess de Nemours, and Colorful Painting. All of them were delivered through air transport with fresh keeping package. The control adopted tissue culture seedlings that have been self-bred in China over the years, such as Louyang Red and Fengdan White.

2.2 Methods

2.2.1Management ofP.suffruticosaseedlings. After receiving the seedlings, we quickly planted them in a 7-9 cm diameter nutrient bowl under shading and moisture conditions, and placed them under the shade frames of the open field or domesticated in a controlled greenhouse until the late of September or early October. After they have become fully survived and grown to a certain size, transplanted them to the field.

2.2.2Construction or selection of shade frames or greenhouses. The first and second experiments were carried out under the shade frames and in the greenhouse. The basic conditions of the shade frames and greenhouse are: ventilation and light transmission, high terrain, clean and hygienic, close to water source, convenient management, strong and firm, and the upper shading yarn and plastic film can be pulled freely, shelter from light and rain, and convenient for people to adjust according to weather conditions. In the first time, the seedlings were placed on the open field of a simple shade frame; in the second time, the seedlings were placed on a 70 cm high wire structure seedbed in a self-controlled greenhouse.

2.2.3Selection and preparation of substrate. The substrate used was prepared by uniformly mixing imported peat and perlite at the ratio of 5∶1. The fiber length of peat was 5-10 mm in the early stage of domestication (i.e. 28-42 d after planting) and was 10-20 mm in the late stage. The principle is to have a high permeability and water permeability without excessive porosity and not moist.

2.2.4Selection of nutrient bowl. In the early stage of planting, namely, 30-60 d after planting, and the mouth diameter was 7-9 cm. When the seedling grew close to or exceeded the diameter, the mouth diameter was 11-14 cm. Both hard and soft plastic could be used, but hard plastic is preferred, because it is easier to place and not easy to deform.

2.2.5Transplanting method. First filled the 1/3-1/2 substrate in the bowl, then gently held the root of the seedlings into the bowl, filled the substrate, and then gently pressed it. The planting depth should follow the principle that the root neck is just exposed after pressing, but not bury the buds.

2.2.6Management methods. Shading and sunshine: after planting, shade 7-10 d under the shading yarn with a shading rate of about 60%. In fine days of summer, shade 60% at 10∶00 to 16∶00 every day. In other times, shading is not necessary.

Watering: 7-14 d after planting, water fully in the first time, and spray water 2-3 times every day. Later, when the surface of the substrate becomes white and dry, water fully again for one time on the principle of seeing moisture and dryness. Except the seedlings planted after 7-10 d, the bowl inside and environment should be kept dry, and the humidity should be kept in the range of 40%-60%.

Ventilation: always keep well ventilated. Usually, it should be kept ventilated every day. When the temperature is too low or too high, even if to maintain a proper temperature, it is required to open the window for 2-3 h every day, especially when both temperature and humidity are high, and there is no natural wind, exchange fan and ventilation fan should be opened for ventilation 1-2 h. However, it should also be noted that the ventilation should not be excessive, to avoid lodging of seedlings.

Fertilization: in addition to the application of 5-10 g of slow-released green manure per pot in the initial substrate, 0.1%-0.3% of compound liquid fertilizer or 1/20 of decomposed cake fertilizer water is applied once every 15-20 d. Disinfection: the entire planting and management process is carried out in a serious and rigorous manner, including the environment, substrate, and tools. The agent used is 600-800 times chlorothalonil solution, 500-600 times carbendazim solution, 3 000 times hymexazol solution, and is sprayed or poured every half month.

2.2.7Change of bowl and field transplantation. After 60-90 d, when the length of the seedling is close to or exceeds the diameter of the seedling, replace with larger bowl (11-14 cm mouth diameter). In the autumn, when the seedlings are fully adapted to the external environment and grow to a certain size, transplant them to the field with soil balls. For some particularly small seedlings, continue to cultivate until the spring of the next year before transplanting to the field.

2.2.8Contents and methods of observation records. Before planting, first observe and make a record of the quality of different varieties of seedlings, such as seedling size, strength, presence and size of top buds, quantity, thickness, length of root systems. After planting, observe the highest and lowest temperature humidity in the environment every day, check the dry and wet conditions in the bowl, the environmental ventilation and the growth of the seedlings, especially the presence or absence of infection of diseases,etc.; observe the record of the number of survived seedlings, deaths, death reason, survival rate, growth potential, plant size, and robustness,etc. In addition, make a record of the daily management details.

2.2.9Control treatment. The tissue culture seedlings domestication methods conventionally used in Luoyang and even related national organizations such as Henan University of Science and Technology, Luoyang National Peony Garden, and Luoyang Academy of Agriculture and Forestry Sciences as a control for comparative analysis. The main differences between such conventional practice and this test method include (i) the seedlings have not undergone a certain low temperature dormancy, (ii) seedling are transplanted together with leaves, (iii) most of the seedlings do not have top buds, (iv) there is no limitation for the transplanting period or season, (v) there is no special requirement for the planting depth, (vi), there is no special regulation on the transplanting bowl (pot) and the change of bowl or pot is basically not necessary during domestication, (vii) ventilation requires less attention, the humidity is greater, and (viii) disinfection is not complete.

3 Results and analysis

3.1 Comparison of domestication and cultivation factors ofP.suffruticosaseedlingsThe quality and status of seedlings and the scientific comprehensive domestication and cultivation measures are the fundamental guarantee for the successful cultivation of tissue culture seedlings. According to the results of this experiment and the control analysis, the strong and well-developed and short stems are the basis for success, and whether there are buds, leaves, when to transplant, the size of bowls or pots, and the depth of planting, and the ventilation are the key factors for the success of domestication and transplanting. Through experiments and comparative analysis, it can be seen that after low temperature dormancy, seedlings with short stems and more than three roots and full buds, after treatment of removing leaves, the seedlings are transplanted in the spring to the appropriate size of nutrient bowls (the mouth diameter of 7-9 cm at early stage, and mouth diameter of 11-14 cm at late stage), the depth should not be excessive to bury the buds, and they should be domesticated and grown for about half a year in a cool, ventilated and hygienic environment. When the seedlings fully survive, the roots are more developed, and the plants reach a certain size, then they can be transplanted to the field. If the seedlings are thin and weak, the roots are few, no buds, have leaves, and transplanted in other seasons, and the bowl or pot is larger, the planting is too shallow or too deep, the environmental humidity is high, the ventilation is poor, and the disease prevention and control is not put in place, then the seedlings will not survive, and domestication will not succeed.

Table 1 Survival of 21 varieties of outdoor domesticated tissue culture seedlings in 2017

VarietynameQtyoftransplantationplantsQtyofsurvivalplantsSurvivalrate∥%BerryGarcia25010240.8Carolina26124593.9CoraLouise19217892.7DuchessdeNemours21216577.8ImpossibleDream25620881.3Lollipop16514688.5MagicalMysteryTour1028482.4LilacPurple29424583.3Oochigeas21817680.7ColorfulPainting1159784.3KopperKettle21317984.0PinkDoodle31726583.6RaggadyAnn1099183.5SmithFamilyYellow745878.4BowlofBeauty4848100.0ScarletHeaven21917881.3BlackSwan807492.5SingingintheRain104104100.0StrawberryCream1015553.9RedDoodle878193.1YellowDoodle27318567.8Total3683296480.5

3.2 Comparison of survival rate between different varieties

From Table 1, it can be seen that for the same facilities and management, but different varieties, the survival rate is quite different. The survival rate of Singing in the Rain, Bowl of Beauty, Carolina, Cora Louise, Red Doodle, and Black Swan is the highest, up to 90%, while the Berry Garcia and Strawberry Cream have low survival rate. In terms of the growth status, the Bowl of Beauty, Red Doodle, Magical Mystery Tour, Singing in the Rain, Smith Family Yellow, Duchess de Nemours, Pink Doodle, and Oochigeas have roubust growth, while the Impossible Dream, Berry Garcia, and Yellow Doodle are weak, the first two varieties have serious leaf spot disease and not suitable for introduction on a large scale, and other varieties are moderate.

3.3 Effects of cultivation facilities and environment on survival rateFrom Table 2, it can be seen that the conditions of the facilities have a great influence on the survival rate of different varieties of tissue culture seedlings. In the multi-span greenhouse with automatic control of temperature, light, humidity and ventilation and with elevated steel wire bed, the domesticated cultivation group has significantly higher survival rate of the seedlings than that of the simple facilities in the open field.

Table 2 Differences in survival rate of different facilities for the same variety

VarietynameSurvivalrate∥%OpenfieldfacilitiesMulti-spanautomaticcontrolgreenhouseBerryGarcia32.2040.80SingingintheRain69.70100.00RedDoodle82.6093.10KopperKettle43.3084.00Lollipop31.7088.50

4 Analysis of survival mechanism

In the long-term evolution and adaptation process of peony or herbaceous peony, certain life cycle habits and rules have been formed. Specifically, they germinate and grow in spring, doze off in summer, develop roots in autumn, and wither and become dormant in winter. In terms of physiology and biochemistry, each period and season has its own division of labor and function. In spring and summer, it mainly carries out nutrient consumption, material synthesis and plant growth. In autumn, it mainly carries out nutrient reserves. In winter, it mainly carries out rehabilitation and material conversion. As to the transplanting season of mature bare root peony plants in the open field, only autumn and winter are most suitable. There is the saying that ifP.suffruticosaseedlingsare transplanted in spring, they will not bloom. In the spring,P.suffruticosasprouts early and consumes more nutrients. If the roots are transplanted at this time, because of the roots are not developed for laying the foundation for absorbing water and nutrients, they will consume much water and nutrient, resulting in imbalance of nutrient supply and consumption, and causing great damage to plants. Consequently, it is difficult for seedlings to recover in the future, so they will not bloom, and even die.

Outdoor domestication and field transplanting of tissue culture seedlings should also follow such annual cycle habits and rules, that is, to simulate natural habitats and situations, otherwise, seedlings will not survive. Although theP.suffruticosaseedlings can be produced on an annual basis in the artificial climate room of the tissue culture room, it is greatly different from the outdoor and the field. The specific mechanism of the successful transplantation of domestication of tissue culture seedlings lies in:

(i) In terms of the transplanting season, the reason for transplanting in the spring, rather than transplanting in autumn and winter, is because the tissue culture seedlings are young seedlings instead of mature plants. If transplanted and domesticated in autumn and winter, firstly, it is necessary to cultivate in the greenhouse in winter; secondly, after cultivation in half a year or so, seedlings have to be transplanted to the field in the spring, and their growth and development stage does not match the natural season, which will break their annual cycle rules. Therefore, the outdoor domestication and transplanting of tissue culture seedlings are only suitable in spring.(ii) Before transplanting outdoors, certain time of dormancy at low temperature is necessary. This is important for laying a certain foundation for nutrient reserve and material transformation. If no such dormancy, it will not effectively provide the energy and nutrients required for plant survival and growth after transplanting to outdoor or the field.(iii) The reason for having top buds but no leaves is that if there is no top bud, there will be no growth point, the seedlings will not continue to grow. If the leaves are not removed, the root system is not well-developed, and the absorption capacity is poor, the water and nutrients necessary for germination and stem and leaf growth cannot be provided in time, leading to imbalance of absorption, supply and consumption, consequently it is not easy for them to survive. This is basically the same as the principle thatP.suffruticosaseedlings are transplanted on Spring Equinox, they will not bloom.(iv) When transplanting, the substrate should not bury the buds, but should even expose the root neck, for the purpose of weakening the respiration and preventing pathogen infection and rot. Because of young buds and root neck of the tissue culture seedlings, they are vulnerable to rot in wet and poor ventilated conditions. (v) At different stages, different size of bowls should be selected. At the early stage, the mouth diameter of bowl should be 7-9 cm; at the late stage, the mouth diameter of bowl should be 11-14 cm because too large or small bowl are not suitable. If the bowl is too large, the substrate will not become dry easily after watering, and it will lead to rot of roots or seedlings. If the bowl is too small, it will not suitable for the rapid growth of the root system and entire plant.(vi) It is necessary to maintain good ventilation. Because the tissue culture seedlings are relatively young, too dry or wet cultivation environment is not conducive to survival, and it is most likely to cause rot of root neck and then death of seedlings due to poor ventilation and high humidity.(vii) The tissue culture seedlings have a higher survival rate in the automatic-control greenhouse than in the simple facilities, which may be mainly because the former is well ventilated, the drainage is fast, the temperature and light are more suitable, the disease is less, and the seedlings are not easy to rot or die.(viii) It has also been found from this experimental study that some varieties of tissue culture seedlings cannot survive, not due to rot of sprouts or roots, but due to germination failure. This is possibly due to insufficient temperature, deep dormancy and no complete break and release. For example, the Impossible Dream and Berry Garcia are like this.

5 Conclusions

Taking thousands of tissue culture seedlings of Itoh peony varieties imported from abroad as test materials, and using the conventionalP.suffruticosadomestication cultivation method as a control, we studied the technical measures and mechanisms for the outdoor domestication of seedlings and the survival of field transplantation. The experimental results indicate that the main factors and measures for effectively protecting the outdoor transplanting of tissue culture seedlings are as follows. (i) Cultivating sound and strong seedlings. The top buds are fully developed, with no less than 3 roots and no less than 3 cm in length. (ii) Transplanting in proper period. Transplanting should be carried out in spring, but not in any time. (iii) It is necessary to select suitable places and facilities for ventilation, drainage and ventilation, and maintain good ventilation. (iv) The substrate should be loose and permeable to water. (v) It is necessary to keep buds and release dormancy before transplantation. (vi) When transplanting, pay attention to the planting depth, the substrate should not bury the buds, but should even expose the root neck. (vii) At different stages, it is necessary to select different size of bowls or pots, and change the bowls or pots according to the size of seedlings. (viii) It should be strictly disinfected and sterilized to prevent infection by bacteria or viruses. (ix) It is necessary to make scientific management of soil, fertility, water, light, air, temperature and humidity and prevention and control of diseases and insect pests. The most important factors or technical measures that are different from the conventional ones and affect survival are (i), (ii), (iii), (v), (vi), (vii). In addition, the survival mechanism was also analyzed, and the reasons and countermeasures for the successful application ofP.suffruticosatissue culture in China were found. Finally, the differences in survival rates between different varieties and facilities were summarized, to provide reference, guidance and promotion for future works.