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VIETNAM ACADEMY OF AGRICULTURAL SCIENCES ***** TONG HOANG HUYEN RESEARCH ON AGRO BIOLOGICAL CHARACTERISTICS AND SOME TECHNICAL MEASURES TO IMPROVE YIELD AND QUALITY OF BO HA KING MANDARIN TREE GROWN I[.]

VIETNAM ACADEMY OF AGRICULTURAL SCIENCES

… *****……

TONG HOANG HUYEN

RESEARCH ON AGRO-BIOLOGICAL CHARACTERISTICS AND SOME TECHNICAL MEASURES TO IMPROVE YIELD AND QUALITY OF BO HA KING MANDARIN

TREE GROWN IN THAI NGUYEN

Major: Crop Science Code: 9 62 01 10

The thesis was completed at:

VIETNAM ACADEMY OF AGRICULTURAL SCIENCE

Supervisors:

1 Prof Dr Ngo Xuan Binh

2 Dr Bui Quang Dang

Ph.D thesis Reviewer 1: Dr Nguyen Van Vuong

Ph.D thesis Reviewer 2: Dr Ngo Hong Binh

The thesis was reported to the Thesis Committee at the Institutional level in the Vietnam

Academy of Agricultural Sciences

At , , 2023

The thesis can be found at the libraries:

1 National Library of Vietnam

2 Library of Vietnam Academy of Agricultural Sciences

3 Library of Fruit and vegetable research institute

LIST OF PUBLISHED SCIENTIFIC WORKS

RELATED TO THE THESIS

1 Tong Hoang Huyen , Nguyen Tien Dung, Nguyen Van Duy, Khoang Lu Pha,

Bui Quang Dang, Ngo Xuan Binh (2022) " Study on the effect of pruning method on growth and yield of Bo Ha king mandarins grown in Thai Nguyen

province ", Journal of Agriculture and Rural Development, Issue 2 May 2022:

pp 9-14

2 Nguyen Tien Dung, Tong Hoang Huyen, Nguyen Van Duy, La Van Hien, Bui

Tri Thuc, Khoang Lu Pha, Bui Quang Dang , Ngo Xuan Binh (2022), " Determination of genetic differences between Bo Ha king mandarin and other

citrus varieties in the North of Vietnam " Vietnam Journal of Science and Technology , No 03 (136) /2022 : No 11-15

3 Tong Hoang Huyen , Nguyen Tien Dung, Nguyen Van Duy, Bui Quang Dang,

Bui Tri Thuc, Nguyen Thi Tinh, Ngo Xuan Binh (2022), " Research on the origin

of the buds in relation to the yield of the Bo Ha King mandarin variety grown in

Thai Nguyen " Vietnam Science and Technology Magazine , Volume 64, February

2022 issue: Pages 34-37

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INTRODUCTION

1 Necessity of the study

The Bo Ha king mandarin tree (Yen The - Bac Giang) has a source origin mount instant with existence face of French since the 19th century According to documents published in the years, 1930-1954 by a number of French agronomists working in Indochina, the Bo Ha king mandarin tree planted by the French has grown well in Yen The area since the 19th century, forming the Bo Ha king mandarin area After peace was established in 1954, Yen The established the Bo Ha king mandarin farm, planting king mandarin varieties for export and domestic consumption The Bo Ha king mandarin was the country's number one orange variety and was famous throughout the region at the national skill competitions held in the ancient capital of Hue, once the pride of the people of the North

However, in the period after the 1980s, the most harmful pest and disease were greening, along with outdated farming techniques, which severely damaged orange production areas, such as Phu Quy orange area - Nghe An, Tuyen Quang- Ha Giang orange areas in general and the Bo Ha king mandarin area ( Bac Giang) in particular The orange growers had to cut down and switch to other agricultural crops, therefore the Bo Ha king mandarin tree was almost only left in the memory of consumers and producers alike

In the period after 2014-2016, the research team of Nong Lam University - Thai Nguyen University investigated and identified some remaining Bo Ha king mandarin trees in farmer households The age of trees were 40- 50 years but most have suffered from greening disease On the basis of investigation, agro-biological characteristics, and application of molecular markers in pedigree determination, it has been determined that these are Bo Ha king mandarin trees, which have different genetic and biological characteristics compared to Ham Yen king mandarin (Tuyen Quang) Simultaneously, the research team has propagated disease-free Bo Ha king mandarin trees, stored and planted at Nong Lam University - Thai Nguyen University The conservation and development of the Bo Ha king mandarin variety is great significance The conservation and development of the Bo Ha king mandarin variety is great significance with production, contribute to the restoration and development of Bo Ha king mandarin, change the structure of plant varieties for agricultural production From the

above practice, the implementation of the topic "Research on agro-biological characteristics and some technical measures to improve yield and quality of Bo

Ha king mandarin tree grown in Thai Nguyen" is necessary and high significance

in science and practice

2 Objectives of the study

Studying some agro-biological characteristics and suitable technical measures to improve the yield and quality of Bo Ha king mandarin, contribute to exploitation, development the valuable gene source in midland and mountainous areas in the North

of Vietnam

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3 The scientific and practical meanings of the thesis

3.1 Scientific meanings

- The results of the study are the premise for future studies about technical measures

to improve the yield and quality of Bo Ha king mandarin fruit

- The research results are the scientific basis to develop the technical process of taking care of the Bo Ha king mandarin tree for the midland and mountainous areas in the North

- The research results of the topic are valuable as documents and materials for teaching and further research on the Bo Ha king mandarin variety in particular and citrus

in particular in the North of Vietnam

3.2 The practical meanings

The results of the study contribute to the conservation, exploitation and development

of genetic resources of Bo Ha king mandarin variety which was almost lost in production,

is an important basis in building the process of taking care of Bo Ha king mandarin tree, including optimization of the technical processes of pruning, propagation by marcotting, fertilizer using and foliar fertilizer application for Bo Ha king mandarin tree

4 New contributions in the thesis

- The thesis researches a systematic assessment of agro-biological characteristics, which is an important reference source for research on orange trees in the Northern Midlands and Mountains regions, and provides arguments, the scientific basis for the building of the intensive care and cultivation process, as well as the planning of planting Bo Ha king mandarin trees in the mountainous provinces of the North

- The research results about bud characteristics, association and origin of buds in

Bo Ha king mandarin is the scientific basis to explain the phenomenon biennial bearing, simultaneously, is the basis to suggest solutions for that phenomenon

- The research results about nutrient supplementation, growth hormones, are the scientific basis for developing solutions to supplement nutrient and growth stimulants

in the production process in order to improve the quality and yield of Bo Ha king mandarin bring high economic efficiency

- Research results on propagation techniques, especially the ability of grafting and rootstock grafting to the growth of Bo Ha king mandarin tree, are an important scientific basis for the use of rootstocks to propagate Bo Ha king mandarin trees

- The results of the study provide a database, which is an important reference in serving the management and development planning of citrus in general, orange trees

in particular, in the Northern Midlands and Mountains region

5 Structure of the thesis

The thesis is presented in 136 pages size A4, including the Introduction (3 pages); Chapter 1 Overview of the literature (25 pages); Chapter 2 Materials, contents, and research methods (13 pages); Chapter 3 Results and discussion (82 pages); Conclusion and recommendations (3 pages); List of 113 documents, including 65 Vietnamese documents, and 48 English documents

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CHAPTER 1 OVERVIEW OF THE LITERATURE

1.1 Some research results on the main agronomical characteristics of citrus

Depending on ecological conditions and propagation form, the age of citrus can

be long or short In citrus orchards that are seeded or propagated by grafting, under favorable conditions, the lifespan can reach a few dozen to more than a hundred years and still give good yields [ 6 3]

Branches of citrus include the main branches which are mother branches, nutrition branches, and fruit branches This classification is according to the function of each type of branch The relationship between branches and shoots is quite close [ 47 ]

1.2 Some research results on technical measures to improve yield and quality of citrus plants

1.2.1 About foliar fertilizer utilization

Foliar fertilizers contain macronutrients, intermediates, and microelements that provide nutrients in a timely manner for plants to grow and develop rapidly Fertilizers containing trace elements, and growth regulators have the effect of increasing the rate of flowering, fruit set, fruit code, and fruit quality and reducing the number of seeds if sprayed at different times with appropriate periods [ 34 ], [ 35 ]

1.2.2 Research on pruning techniques to shape citrus trees

Technical art cut trim gives tree yes abs including the internal content main after: Pruning to shape, pruning after harvest, pruning flowers, pruning fruit, and renovating old and weak trees The main types of pruning and shaping usually applied are spherical

or hemispherical cutting, but currently, most countries with a developed citrus industry (USA, Australia, Israel, Taiwan, China ) has been gradually transitioning to a Y-shaped pattern (initiating style, jar or open heart type .) even in a fan-less style to facilitate harvesting by machine Pruning usually works better when it is done in concert with other techniques such as fertilizer, watering, and pest control Cut trim can not usually present clearly effect in the early years of pruning [23]

The removal of flowers, malformed fruits, clusters of flowers, and fruits that are too thick will contribute to reducing the above imbalance According to the author Vu Cong Hau, (1996 ) [21], pruning is more and more widely applied The rule is to cut carefully when the tree is young, cut less when the tree is old, cut more in winter or drying seasons , when the tree in stage which cease born chief cut much, grow strongly cut little

1.2.3 Research on technical measures of bark grafting method for citrus trees

Circling the bark has two main effects: promoting flowering and increasing fruit setting rate Therefore, it is possible to apply this technique to the Bo Ha king mandarin variety to promote flowering as well as increase the fruit setting rate and fruit yield of this orange variety

1.3 Some research results on citrus grafting techniques

Nowadays, it is possible to graft in vitro (micrograft) to create disease-free plants

Microshoot tip grafting was first applied by Murashige in 1972 It was then further improved by Navarro in 1975, 1976, 1980, and 1981 and Hong Ji Su in 1984 The technique of micrografting of meristem includes stages: preparation of the rootstock,

preparation of the shoot apexs, grow plants in vitro by micrografting, and then planting

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in pots [38]

There are many trees that are used as rootstocks for citrus trees In the South, honey oranges are used, in the North use pomelo, recently tangerine and some new rootstocks are used

According to the research results of Khuat Huu Trung in 2022 [39], the effect of the grafting season of Tay Giang orange trees in Quang Nam When using the eye grafting

method on rootstock of Aurantii Fructus (Citrus aurantium) in Thai Binh, it shows that

the most suitable time to graft is in February, then is in the mid-August, and the lowest

is in mid-October, expressed through survival and germination rate of grafts, growth parameters including length and width of leaves, length, and diameter of grafted branches Bui Quang Dang et al in 2021 [12], when performing eye grafting of Xa Doai and

CS1 oranges on the rootstocks of Aurantii Fructus in the North Central region, it was

found that the most suitable time to graft is Summer (June), then Autumn ( August), and the lowest is in the winter season (mid-December) The rate of germination the first time (after 30 days) and the rate of germination the second time (after 100 days) when grafted in the summer crop reached 80.0% and 82.5% Regarding

mid-to the growth of grafted trees, Xa Doai grafted orange trees’s height is about 72.5 cm The diameter of the rootstock is 0.91 cm The length of the graft is 45.7 cm The diameter of grafted branches is 0.71cm; The number of level 1 branches is about 2-3 branches; and CS1 grafted orange tree’s height of 75.7 cm; rootstock’s diameter is 0.92 cm; length of grafted branches 47.2 cm; the diameter of grafted branches 0.72 cm, the number of level 1 branches is 2-3 branches

CHAPTER 2 RESEARCH MATERIALS, CONTENTS, AND METHODS

2.1 Research site and materials

2.1.1 Research site

Research location at University of Agriculture and Forestry, Thai Nguyen

University, Quyet Thang Commune, Thai Nguyen City, Thai Nguyen Province

2.1.2 Research objects and materials

- Research objects: 4 years old Bo Ha king mandarins; Planting density is about 400-800 trees/ha

- Control: Ham Yen king mandarin variety ( provided by Ham Yen Fruit Seed Center, Tuyen Quang province )

2.1.3 Research time

The study period was conducted from 2017 to 2020

2.2 Research contents

2.2.1 Research on some agro-biological characteristics of Bo Ha king mandarin variety

- Research on morphological characteristics of Bo Ha king mandarin variety: Stems, branches, leaves, flower, fruit, ability to give yield and quality of the Bo Ha king mandarin fruit

- Research on genetic diversity of Bo Ha king mandarins by molecular markers

- Research on the phylogenetic origin and the relationship between the buds in the Bo Ha king mandarin tree

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- Research on the growth characteristics of the buds and the correlation between the growth of fruit branches and fruit yield in the Bo Ha king mandarin variety

2.2.2 Research on the influence of some technical measures on growth, yield, and quality of the Bo Ha king mandarin

- Research on the effect of pruning on growth and yield of the Bo Ha king mandarin tree

- Research on the effect of bark grafting method on yield and quality of the Bo Ha king mandarin tree

- Research on the effects of some foliar inoculants products on the yield and quality of

Bo Ha king mandarins

- Research on the effects of some foliar inoculants products combined with pruning

on the yield and quality of the Bo Ha king mandarin

- Research on the influence of rootstock and grafting time on the growth of the Bo Ha king mandarin

2.3 Research methods

2.3.1 Research methods on some agro-biological characteristics of Bo Ha king mandarin variety

2.3.1.1 Research on morphological characteristics of the Bo Ha king mandarin

The research was conducted on a 4-year-old Bo Ha king mandarin garden, randomly selected 9 trees to do experiments, 3 times repeated Experimental plants were evaluated for canopy form, morphological characteristics of stems, leaves, flowers, fruits

* Targets and tracking methods:

- Evaluation of shape canopy

- Stem diameter (cm): Measure with Palmer ruler at the point which is ten cm from the ground

- Tree height (cm): Measured with a long ruler from the ground and measured to the highest point of the canopy tree

- Canopy diameter (cm): Measured with a tape measure, measure the tree

canopy’s projection in the orientation from East-West and South-North directions, then take the average value jar

- Number of branch levels: Count the number of levels of branches currently on the tree

- Thorn’s Density

- Measure leaf’s length and width (mm)

- Measure the length and width of the leaf’s waist (mm) (if)

- Characteristics about flowering, fruiting, and yielding capacity:

- Fruit ripening period: When there are more than 20% of the fruits ripe

- Theoretical yield/tree (kg/tree): Number of fruits/tree × weight of fruit

(g/plant)

- Actual yield: Weigh the total number of fruits of each tree from which to

calculate the average yield (kg/tree)

- Average fruit weight, edible portion (g/fruit)

- Composition analysis of Fruit: Measure Brix, Total Sugar (%), Total Acid ( % ),

Vitamin C (mg/100g fresh fruit)

2.3.1.2 Research on genetic diversity of Bo Ha king mandarins by molecular

markers

The research was conducted on 32 citrus samples including 4 samples of Bo Ha king mandarins, 1 sample of Bo Ha citrus, 11 samples of Ham Yen king mandarins, and some other citrus varieties being grown in the North of Vietnam

- The primers used in the study were designed based on the author's research Oliveira et

al (2010) [89],

* Research methods

Genetic diversity analysis of some local citrus varieties in Vietnam by RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat) markers

- Extraction of total DNA:

- RAPD and ISSR reactions were conducted with random primers according to

Malik's method [88] ( Malik et al , 2012)

* Targets and tracking methods:

- The shoots growing from the experimental branches are marked with dates so that the growth criteria can be followed

- Number of batches to track: Spring crop, Summer crop, Autumn crop, Winter crop

- The growing time is counted from the time of buds formation to the mature branches in Spring, Summer, Autumn, and Winter, and compared

- The growth behavior of Spring, Summer, Autumn, and Winter crops are tracked and compared Measure the length of the bud once every 7 days Measure until the length of the buds does not change at the last 3 measurements, then it is considered that the buds have stopped growing in length at the first measurement of the last 3 measurements The bud is called mature when it no longer increases in length and the young leaves have turned dark green

- Determine the number of leaf nodes and the number of mature

2.3.1.4 Research on the correlation between fruit branch growth and fruit yield in the

Bo Ha king mandarin tree

* Experimental setup:

In a 4-year-old orchard, 10 plants were randomly selected for the experiment The uniform experimental plants represent the normal growth of the plants in the experimental garden, the plants are not damaged by pests and diseases On every tree choose from 4 horizontal branches spread evenly on 4 sides - branches over 1-year-old Select a branch with a diameter of about ≥ 1.0 cm, mark the branch close to the main stem, and monitor the ability to produce buds, the growth of the mother branch, the age of the mother branch in the marked part or more, and when the branch has buds, proceed to mark the buds which clearly state the date of the buds, and continuously monitor the buds growing on the experimental branches throughout the experimental period The total number of experimental branches initially monitored was 40 branches On the basis of the branches marked in 1 year, to monitor the growth criteria of the fruit branches and fruit yield, on that basis, use mathematics to analyze the correlation between the biological criterias of branch growth and fruit yield

* Criteias and methods according to track:

The growth criterias of fruit branches include length, diameter, and the number

of leaves The above parameters were measured separately on experimental branches The yield parameters of fruiting branches were also measured separately for each fruit-bearing branch The above data will be analyzed to determine the relationship between fruit tree growth and yield capacity

2.3.1.4 Research on the phylogenetic origin and the relationship between the buds in the Bo Ha king mandarin tree

* Experimental setup:

The experimental garden was 4 years old and randomly selected 10 trees for monitoring and evaluation (according to the method of Ngo Xuan Binh et al in 1998 ) The shoots on the monitored branches were observed and recorded continuously for

3 years (2019, 2020, and 2021)

* Tracking criterias:

The number of buds in a year, and the rate of branches forming according to the season The origin of the buds formation of the year Determine the origin of the mother branch (the branch that produces the fruit branch) and the origin of the fruit branch (the branch that produces flowers and fruits) Determine the relationship between the buds formations of the year

Formula 1: Pruning according to the process of the Fruit and Vegetable Research

Institute: Cut trim with 3 batch main: the Spring crop (January to February, combined with pruning flowers), the Summer crop (April to June, combined with pruning young fruit) and the autumn crop (August to September, only pruning branches)

Formula 2: Pruning according to the initiation style: pruning level 1 or level 2

branches in the middle of the canopy, leaving 3-5 main branches Regularly cut off branches that tend to grow tall, diseased branches and branches in the canopy that have a small diameter, toothpick branches

Formula 3: Control: No pruning

* Criterias and tracking methods:

- The rate of buds on all kinds of branches; The growth of the buds; Flowering time and fruiting rate; Factors of productivity

2.3.2.2 Research on the effect of the rinding method on yield and quality of Bo Ha king mandarins

* Experimental design:

The experiment was conducted with 5 formulas:

Formula 1:Control: No shelling

Formula 2: Rinding level 1 branches on October 30 every year; Formula 3: Rinding level 1 branches on November 15 every year; Formula 4: Rinding level 1 branches on November 30 every year; Formula 5: Rinding Level 1 branches on December 15 every year

* Criterias and tracking methods:

Flowering time, bloom, and end bloom; Fruit set rate; Yield affecting factors

2.3.2.3 Research methods on the effects of some foliar fertilization inoculants on yield and quality of Bo Ha king mandarin

- Experimental design: The experiment was arranged in a randomized design, on a 4-year-old Bo Ha king mandarin garden, the experiment was repeated 3 times

- Experimental formulas with some foliar fertilization inoculants

+ Formula 1: Control: Spraying with water

+ Formula 2: Spray inoculant 1 (Bo: 2,500 mg/l, add Ca, Cu, Fe, Mg, Mn, Zn,

and special additives for plants)

+ Formula 3: Spray inoculant 2 (Bo: 2,600 mg/l, supplemented with Ca, Fe,

Mg, Cu, Mn, and special additives, just enough 100%)

+ Formula 4 : Spray inoculant 3 (N: 5%, P 2 O 5 : 5%, K 2 O: 5%, Bo: 100 ppm, Zn: 200 ppm, Cu: 100 ppm, Fe: 50 ppm, Mn: 100 ppm)

The formulations were carried out under the same growing and care conditions

- Spray inoculants in the following periods: Spray 1: spray 10 days before

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flowers bloom, Spray 2: spray when flowers are in full bloom, Spray 3: spray 10 days after flowers bloom The way to spray: spray wet all flower clusters, and flower buds

at the end of the afternoon session

* Criterias and tracking methods: Fruiting rate; Number of fruits/tree; Fruit weight (kg); Yield/plant (kg); Fruit quality criterias

2.3.2.3 Research methods on the effects of some foliar fertilization inoculants in combination with pruning on the yield and quality of Bo Ha king mandarin

* Select plants for the experiments: The tree is 4 years old, is at the stage of business, and is most vigorous, choose trees that are relatively uniform in terms of initial growth and development The formulations were studied under the same growing and growing conditions

* Experimental design: The experiment was arranged in a fully random design

on an orange garden which is from 4 years old, with 4 treatments, 3 replicates, and 3 trees each time with different treatments are as follow:

+ Formula 1: Control: Water spray combined with the best pruning method + Formula 1: Spray inoculant 1 in combination with the best pruning method + Formula 2: Spray inoculant 2 combined with the best pruning method

+ Formula 3: Spray inoculant 3 in combination with the best pruning method The formulations were carried out under the same growing and care conditions Only for the best pruning method, all the recipes were pruned according to the method

of the Fruit and Vegetable Research Institute in the year with good number of fruits and all recipes are pruned according to the initiation method in little fruit year

- Method of spraying inoculants in different periods: Spray 10 days before flowers bloom, spray when flowers are in full bloom, and spray 10 days after flowers bloom The way to spray: spray wet all flower clusters, and flower buds at the end of the session afternoon

* Monitoring criterias : Fruiting rate and yield factors, fruit quality criterias

2.3.2.4 Research on the influence of fertilizer to yield and fruit quality criterias of Bo Ha king mandarin

- Research on the effect of the dose of potassium fertilizer on the yield of Bo Ha king mandarin tree

The experiment was arranged in a fully random design with 7 treatments, 3 replicates, and 3 trees each time are as follow:

Formula 1: Control- manure; Formula 2: 0 kg potassium chloride; Formula 3: 0,2 kg potassium chloride; Formula 4: 0,4 kg potassium chloride; Formula 5: 0,6 kg potassium chloride; Formula 6: 0,8 kg potassium chloride; Formula 2: 1

Formula 1: Using rootstock is a Citrus hystrix tree; Formula 2: Using rootstock

is sour pomelo; Formula 3: Using the rootstock is Dien pomelo

Grafting method used: eye grafting

* Targets to track:

- Evaluation of the ability of grafted eyes to adapt and grow on

rootstocks

- Evaluation of pests and diseases

- Determine the percentage of grafted plants that pass and do not meet the

standards for garden release

2.3.3 Data processing methods

The aggregated data are processed by statistical processing software: IRISART 4.0 and Microsoft Excel

CHAPTER 3 RESEARCH RESULTS AND DISCUSSION

3.1 3.1 Research results on some agro-biological characteristics of Bo Ha king mandarin

3.1.1 Research results on morphological characteristics of Bo Ha king mandarin

Bo Ha king mandarins tree has a relatively good growth ability, 4 years old trees have the height from 212.13 cm to 229.27 cm, with an average of 218.66 cm The tree branch is inclined, there are almost no thorns on the branches The root diameter ranges from 4.88 cm to 5.23 cm, with an average of 5.10 cm; Foliage is hemispherical, canopy diameter ranges from 176.72 cm to 203.20 cm, averaging 189.26 The tree has a large branching ability, the number of level I branches is from 3.00 to 3.83, the average is 3.44 branches, the average diameter is 3.26 cm, the average height of branching is 15.60 cm The number of level II branches ranges from 12.60 to 15.74 branches, with

an average of 13.87 branches The ratio of level II branches / level I branches ranged from 3.89-4.20, with an average of 4.04 Comparing the morphological characteristics

of the Bo Ha king mandarin tree with that of the Ham Yen king mandarin tree in the