Breeding and Application Process of a New Variety ‘Dianyinhua 3’ in Lonicera macranthoides Hand.-Mazz.

Shaobo ZHANG Xiaofei YANG Zecheng LI Jinqiao PENG Rongjiao DONG Tianming WANG Quan LIANG

Abstract ‘Dianyinhua 3 is bred through the selection of a good single variant plant based on Hunan Lonicerae Flos variety ‘Xianglei 1 as the basic material. The scions of the variant plant were grafted and propagated with wild Lonicera macranthoides Hand.-Mazz. as rootstocks. This variety belongs to L. macaroides， which has the characteristics of early flowering， large and numerous buds， concentrated flowering and abundant yield. The content of chlorogenic acid is as high as 6%， and the content of luteolin is 0.017%. It matures from late May to early June in Yunnan. The buds are rod-shaped， slightly curved， 4.6-5.8 cm long， with a diameter of 6.47 mm at the top and 2.68 mm at the bottom， and the petals have a thickness of 0.33 mm. The thousand-flower bud weight was 15.8-18.2 g， and the average yield of dried flowers per plant was 1 360-1 680 g in the peak period， and the yield was 4 887 kg/hm2. It is suitable for the introduction and trial planting in Xiangyang Mountainous Area or rocky desertification mountainous area in Yunnan， with an altitude lower than 2 200 m or in areas with similar climates such as Sichuan， Guizhou and Guangxi.

Key words Lonicera macranthoides Hand.-Mazz.; Dianyinhua 3; Variety breeding

Lonicerae Flos refers to the dried flower buds or open flowers of Lonicera macranthoides Hand.-Mazz.， L. hypoglauca Miq.， L. confusa DC.， or L. fulvotomentosa Hsu et S.C.Cheng[1]. It was selected in the "Thirteenth Five-Year" planning textbook Medicine Plant Breeding of Peoples Medical Publishing House in 2018. Lonicerae Flos contains chlorogenic acid， volatile oil， flavonoids， triterpene saponins and other components[2-3]， has the functions of clearing away heat and toxic materials and dispelling wind and heat， and is mainly used for the treatment of carbuncles， paralysis， erysipelas， wind-heat cold， fever of warm disease， and liver protection and anti-cancer activity[4-6]. In addition to being used in pharmaceuticals， the use is becoming more and more extensive， such as daily chemical， beverage， beauty， health care products， etc.， and the amount is also increasing[7]. Meanwhile， Lonicerae Flos also has the functions of preventing wind and fixing sand， maintaining soil erosion， improving soil and beautifying cities. Therefore， it has a broad market and huge business opportunities[8].

Yunnan is one of the main producing areas of Lonicerae Flos， and its resource reserves are large. Since 2010， Lonicerae Flos has been introduced from Shandong and other places. Due to the small amount of flowers， low yield， and short time from budding to flowering （about 7 d）， the promotion progress is slow. Over the years， it has mainly relied on the collection of wild resources to supply the market. No systematic evaluation and selection has been conducted on the germplasm resources of Lonicerae Flos， and there have been no research reports on the breeding of new varieties. The new Lonicerae Flos variety ‘Dianyinhua 3 was selected by Kunming Yingwu Agricultural Technology Co.， Ltd. with a good single variant plant of Hunan Lonicerae Flos variety ‘Xianglei 1 as scions and the wild Lonicerae Flos in Kunming， Yunnan as rootstocks for grafting. Since 2017， this variety has been demonstrated in Shiping County， Shizong County， Yiliang County， Huaning County， Zhenyuan County， Lancang County， Funing County， Yanshan County and other places， with a planting area of nearly 700 hm2.

Breeding Methods

Original material

Lonicerae Flos ‘Dianyinhua 3 was bred from a good single variant plant of Hunan Lonicerae Flos variety ‘Xianglei 1， belonging to L. macranthoides Hand.-Mazz.

Breeding process and method

In 2008， the seeds of the Lonicerae Flos variety ‘Xianlei 1 were sown at the base of Goujie Town， Yiliang County， Yunnan Province， and about 800 seedlings were grown. In 2010， the seedlings were transplanted and 622 plants survived. A variant plant was found in the field in 2012. It has narrowly ovate young leaves， the leaf surface and petiole of which had sparse pubescence. The young leaves are yellowish green， with light rust red leaf surface. The mature leaves are larger， green， and the pubescence on the leaf surface and petiole disappear. The flower buds do not bloom or rarely flower when they grow up， and are neat. The flower buds are greenish green at the beginning， and then turn yellowish white. The plant was named ‘Dianyinhua 3.

From 2013 to 2017， the seedlings were propagated by the grafting method using the roots of the wild Lonicerae Flos in Yunnan as rootstocks and the tender shoots of ‘Dianyinhua 3 as scions. In March 2014， grafting was performed for raising seedlings， and the roots of wild Lonicerae Flos of about 12 cm long as were selected as rootstocks， which were cut with a knife at the grafting site longitudinally by 1 cm long at the cambium; the branches of the variant plant were cut into 2 cm long segments as scions， each of which had at least one leaf axillary bud， the lower end of which was cut off by 1 cm from the phloem and other parts except the cambium; and the above rootstocks and scions were bound with plastic tape. After grafting， the plants were cultivated in a greenhouse in nutrition bowls， or directly planted in a seedling bed. In September of the same year， they were planted at Yiliang base at a row spacing of 1.4 m×1.3 m. A variety comparison test was conducted with ‘Xianglei 1 as a control check （CK）.

Characteristics

Botanical characteristics

The new Lonicerae Flos variety ‘Dianyinhua 3 belongs to L. macranthoides Hand.-Mazz. It is a perennial liana. The stems and branches are thick， the leaves are thick and dark green， and the internodes are short. There are few branches growing excessively and many flower branches.

Stems and branches

‘Dianyinhua 3 Lonicerae Flos is a kind of evergreen shrub， which has developed root system， good rooting ability and water retention capacity， so it has strong adaptability to the growth environment， and can be cultivated in mountainous areas， hills and flat fields and grow well in clay， sand， acid and alkaline soils. It has thick and strong stems and branches and cylindrical and thick stems. The skin of the old stems flakes longitudinally， and the axillary buds have good germination. The plants have many branches， and the plant type is compact and dense， which is conducive to pruning into a reasonable tree structure. The average length of internodes is （6.85±2.67） cm. The flowering branches of 2-year-old plants accounts for 93.5%， and the proportion of flowering branches of 3-year-old plants reaches 99.8%. There are almost no long branches growing excessively， and the flower buds occupy the top. The height of three-year-old plants is 1.4-2.0 m， the main stem diameter is 3-4 cm， and the crown is about 4 m2. This morphological characteristic is the basis for creating a reasonable plant morphological structure， increasing the number of flowers， and achieving high yield and efficiency.

Leaves

The young leaves of ‘Dianyinhua 3 has a narrow oval leaf shape， a blunt leaf tip， a truncated base， sparse pubescence on the leaf surface and petiole， yellowish green leaves and light rust red leaf surface， while mature leaves are larger and green in color， and the pubescence on leaf surface and petiole disappear. The average length of the leaves is （10.17±3.25） cm， and the width is （4.68±2.55） cm. These leaf characteristics are conducive to the formation of a larger photosynthetic area and higher photosynthetic efficiency， and increase the accumulation of photosynthetic products and dry matter， which are the high-yield morphology and physiological basis of this new variety.

Tomenta

The young leaves and stems of ‘Dianyinhua 3 Lonicerae Flos have short tomenta. As the growth process progresses， the tomenta gradually disappear.

Flowers

The total pedicels are clustered， and the buds are dense， stick-shaped and slightly curved， 4.6-5.8 cm in length， 2.68 mm in the diameter of the base of the buds， 6.47 mm in the diameter of the top of the buds， and 0.33 mm in the thickness of the petals. Its anther length， width， stigma diameter and nectary diameter are larger. The surface of the buds is bluish green or yellowish green. The flower buds do not bloom or rarely bloom when they grow up， and the corolla lobes of the open flowers are less than half of the full length. The flower buds are neat. The flower buds are greenish-green at the beginning， greenish-yellow 10 d later， began to wither 14 d later， and do not bloom until withering. However， the flower buds of L. japonica Thunb. bloom 3-4 d after the flower buds grow large and wither the next day after flowering. The flower buds of ‘Dianyinhua 3 are slightly hard， but slightly flexible when pinched with fingers. They are fragrant and slightly bitter in taste.

Economic traits

Thousand-bud weight

The ‘Dianyinhua 3 Lonicerae Flos has a thousand-flower bud weight of （312.3±14.54） g， which is slightly higher that of ‘Xianglei 1， and 159.4% higher than the dry weight of L. japonica [（120.4±12.46） g].

Bud number

‘Dianyinhua 3 has fewer excessively-growing branches and more flowering branches. The 1-2 year old plants have a few excessively-growing branches， and there are nearly no excessively-growing branches after the third year. The number of flower buds per plant in the 2-year-old plants is 5 974， which is 7.7% more than that of ‘Xianglei 1 （5 546） and 45.53% more than L. japonica （4 105）.

Demonstration yield

In 2018 and 2019， the yield was measured in Zhushan Town and Goujie Town of Yiliang County and Kuishan Town of Shizong County in Yunnan Province. The average yield of dried flowers per hectare in ‘Dianyinhua 3 Lonicerae Flos was 4 887 kg per hectare， which increased by 594 kg of dried flower per hectare， accounting for 13.84%， compared with "Xianglei 1" （average yield of dried flowers per hectare 4 293 kg）， and was 2.72 times that of Shandong L. japonica （1 800 kg/hm2）.

Medicinal active ingredient contents

For ‘Dianyinhua 3 Lonicerae Flos， galuteolin and chlorogenic acid are the main active ingredients. Tested by Yunnan Yunce Quality Inspection Co.， Ltd. in 2019， the content of luteolin in "Dianyinhua 3" Lonicerae Flos reached 0.017%， and the content of chlorogenic acid reached 6.0%， which was 1.73 times higher than that of ordinary Lonicerae Flos （2.2%）， and was 3 times of the minimum index （2.0%） specified in the 2015 edition of Chinese Pharmacopoeia.

Adaptability and resistance

‘Dianyinhua 3 Lonicerae Flos is resistant to drought， waterlogging and diseases and pests. According to the comprehensive agricultural technology development center in Eshan County， Yunnan Province and Linxiang Plant Protection and Plant Inspection Station in Lincang City， ‘Dianyinhua 3 is highly resistant to powdery mildew. It is not only suitable for planting in mountainous hills， but also suitable for developing in dam areas with fertile soil. It is also an excellent plant for wind protection and sand fixation， water and soil conservation， and greening. It has strong anti-pollution ability， rich and pleasant floral fragrance， and high ornamental value and environmental protection effect， and it is also an excellent plant for urban greening.

Key points of the cultivation techniques

Site selection and field preparation

It can be planted on flat or sloped land with sufficient sunlight. For hole planting， holes are dug according to the row plant spacing of 1.8 m×1.8 m or 1.8 m×2.0 m. The depth of the hole is 0.4 m and the width is 0.4 m， and 10 kg of decomposed organic fertilizer is applied as the base fertilizer per hole. Supports such as wooden poles or cement poles are set next to the seedlings or large stones are used as climbing objects.

Field management

Intertillage and weeding are performed 4-5 times a year to keep no weeds growing around the plants. The first time is before growth of spring shoots in spring， and the last time is in winter. Intertillage and weeding should be performed from outside to inside， and the depth of the outside is shallow， so as not to damage the roots.

Topdressing is performed 3 times a year， respectively in spring， summer and winter. In early spring from February to March， each plant is applied with compost organic water fertilizer 10-20 kg and superphosphate 50 g， to promote the growth of flower buds; from April to June after harvest， each plant is applied with compost organic water fertilizer 10-20 kg or ternary compound fertilizer 10-15 g to restore the growth of the plant; from November to December， each hole is applied with compost or manure fertilizer 5-10 kg， superphosphate 100 g， and ammonium sulfate 50 g by digging a circular ditch， applying the fertilizers and ridging， so as to promote the growth of autumn shoots， and lay a foundation for high yields the following year.

Pruning

After planting， an auxiliary pole （wooden pole or cement pole， etc.） with a height of 1.3-1.6 m is set up next to each plant， and bound with a seedling， which grows up along the pole， and when the plant is flush with the auxiliary pole， the stem tip is removed to promote branch growth and cultivate the main stem. Pruning and shaping is performed 2 times a year， during which the branches falling to the ground are cut off， and the upward or horizontal branches are cut to make the plant upright. If a rock is used as a climbing object， the branches should be pulled according to the shape of the climbing object to make a reasonable distribution. Every autumn and winter， the weak， diseased， withered， old， dense and long branches of the plants are cut off.

Control of diseases and pests

The diseases and pests are mainly Semiaphis heraclei （Takahashi） and powdery mildew.

Powdery mildew mainly damages leaves， sometimes stems and flowers. The disease spots on the leaves are white dots at the beginning， and then expand into white powdery spots， and later the entire leaf is covered with a white powder layer. In severe cases， the leaves yellow or even fall off.  The disease spots on the stems are brown， irregularly shaped， with white powder on them. The flowers are twisted and fall off in severe cases. Prevention method： We should pay attention to pruning， increase ventilation and light transmission. At the initial stage of the disease， it can be controlled by spraying 50% colloidal sulfur 100 g in water 20 kg or 2 000 times dilution of 15% triadimefon WP.

S. heraclei damages the leaves and young shoots， causing the leaves and flower buds to curl， stop growth and the yield to drop sharply. The pest is more severe from March to June， especially in rainy days， and spread faster. Control method： The pest is controlled by spraying 2 500-3 000 times dilution of 0.36% matrine aqueous solution and 1 000-1 200 times dilution of 1% azadirachtin matrine emulsifiable concentrate continuously for several times until it is killed.

Shaobo ZHANG et al. Breeding and Application Process of a New Variety ‘Dianyinhua 3 in Lonicera macranthoides Hand.-Mazz.

Propagation Techniques

The breeding methods of elite seedlings of L. macranthoides mainly include direct seeding， grafting， cutting and tissue culture. It takes a long time from sowing to flowering for direct seeding， and a long time. Some rootstocks carry viruses， fungi and bacteria， leading to serious diseases such as root rot， short life span （about 5 years）， low survival rate and low affinity of grafted seedlings. Cutting seedlings are difficult to root， and the survival rate is low （it is difficult to exceed 10%）. Tissue culture seedlings are truly high-quality fungus-free and bacteria-free virus-free seedlings， but the production is difficult. Under normal tissue culture production management， the survival rate is difficult to exceed 10%. Even if corresponding special measures are taken， the survival rate is difficult to exceed 50%.

Based on this， our research team explored different grafting methods of ‘Dianyinhua 3 Lonicerae Flos. The results showed that using the roots of wild Lonicerae Flos as rootstocks for grafting seedlings achieved the best effect， and the survival rate was more than 60%. The seedling production cost was between 4.5 and 6.0 yuan/plant， and was significantly higher than the production cost of L. japonica cutting seedlings.

Using L. japonica as rootstocks

With the one-year-old cutting seedlings of L. japonica as rootstocks， they are cut with a knife at the grafting site longitudinally by 1 cm long at the cambium; the branches of the variant plant are cut into 2 cm long segments as scions， each of which has at least one leaf axillary bud， the lower end of which is cut off by 1 cm from the phloem and other parts except the cambium; and the above rootstocks and scions are bound with plastic tape. After grafting， the plants are cultivated in a greenhouse in nutrition bowls， or directly planted in a seedling bed. We should do a good job of fertilization and weeding management in the nursery， and transplant the seedlings around the next spring. Viewed from the outside， these grafted seedlings are exactly the same as the natural variant plant of ‘Xianglei 1， and there are no differences in plant morphology and flower buds between the two.

Using shoots of grafted seedlings as scions

The new shoots grown from the grafted seedlings cultivated as above are used as scions， and the adult plants of local Lonicerae Flos， that is， the adult plant of L. macranthoides， are used as rootstocks to carry out high-level grafting. When the new shoots growing out in the very year is 72-84 cm in height， the plants are topped， and flower buds grow out. Meanwhile， the grafted seedlings cultivated in the previous year are transplanted in the field， and about 50% of the grafted seedlings are hung for flowers that year. From the appearance， these new shoots， the grafted seedlings cultivated in the previous year and the field transplanted seedlings of the grafted seedlings are exactly the same as the natural variant plant of L. macranthoides， and there are no differences in the plant morphology and flower buds between the four.

Cutting propagation

First， 1-2-year-old robust branches are selected and cut into cuttings of about 25 m long， each with 2 to 3 nodes. The seedling bed is ditched according to a row spacing of 25 cm and a depth of 15 m. The cuttings are placed obliquely into the ditches every 3-5 cm， and covered with soil firmly， and the cuttings are exposed to the soil surface by about 1/3. The cuttings are placed while ditching， and the buried cutting are then watered and shaded （shading degree 70%-80%）.

Using the root system of wild Lonicerae Flos as rootstocks

The roots of wild Lonicerae Flos of about 12 cm long are selected as rootstocks， which are cut with a knife at the grafting site longitudinally by 1 cm long at the cambium; the branches of the variant plant of ‘Xianglei 1 are cut into 2 cm long segments as scions， each of which has at least one leaf axillary bud. The scion treatment and grafting method are the same as before， and provisonal planting can be planted in a nutritious bowl or directly on the seedling bed. Through exploration and comparison， it was found that Kunming Lonicerae Flos has the best effect as a rootstock. The grafting can be carried out indoors regardless of day and night and location， which reduces labor intensity and greatly improves efficiency. The field observation showed a high survival rate and a long harvest time of adult plants （some rootstocks have a short life span）. Both the scions and rootstocks are Lonicerae Flos， so the quality is not affected after grafting.

Promotion Prospects

‘Dianyinhua 3 has the characteristics of strong growth potential， concentrated flowering， long flowering period， high yield， good quality， strong resistance， and easy picking. It is suitable for Yunnan areas with an altitude lower than 2 200 m， or introduction and trial planting in sunny hills or rocky mountains in Guiyang， Sichuan， Guangxi and other places with similar ecological conditions， and has broad prospects for promotion and application.

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