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One of the proposed solutions is to select suitable rubber varieties good growth, high yield and cold tolerance, accompanying with breeding and cultivation techniques to develop rubber i

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MINISTRY OF EDUCATION

AND TRANING

MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT

VIETNAM ACADEMY OF AGRICULTURE SCIENCES

NGUYEN XUAN TRUONG

RESEARCH ON TECHNICAL MEASURES TO DEVELOP RUBBER TREES IN THE NORTHERN

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The project is completed at VIETNAM ACADEMY OF AGRICULTURE SCIENCES

Scientific superviors: 1 A Dr Nguyen Van Toan

At h date month year 2020

This dissertation can be found at

- National Library

- The Vietnam Academy of Agriculture Sciences’ Library

- The Northern Mountainous Agriculture and Forestry Science Institute’s Library

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LIST OF PUBLICATIONS RELATED TO THE THESIS

1 Nguyen Xuan Truong, Nguyen Van Toan (2019), "Research results on the selection of some high-yielding rubber lines in the

Northern mountainous areas", Vietnam Journal of Agricultural

Science and Technology, 9 (106), p 39-43

2 Nguyen Xuan Truong, Nguyen Van Toan, Le Van Duc, Bui Thi

Hai Yen, Nguyen Khanh Linh and Cao Phi Bang (2018), “Effects

of low temperatures on some physiological and biochemical

reactions of five varieties rubber (Hevea brasiliensis)”, Scientific

report on biological research and teaching in Vietnam - The 3rd National science conference

3 Cao Phi Bang 1* , Nguyen Xuan Truong2*, Nguyen Van Toan2, Le Van Duc3, Le Manh Tu4, Nguyen Thi Hai Yen5 (2017), "Influence

of potassium on some physiological and biochemical indicators of

rubber trees (Hevea brasiliensis) under short-term low

temperature shock”, Journal of Agriculture and Rural Development No 24, p 41-46, Periodical 2, December 2017

4 Nguyen Van Toan, Dao Ba Yen, Nguyen Xuan Truong, Le Thi Trang and Nguyen Thi Thu Cuc (2016), TBKT01-24: 2016/BNNPTNT: Technical procedure for rubber breeding in the Northern mountainous region, Decision No 62 / QD-TT-CCN dated 3 March, 2016 of the Director of the Crop Division, MARD

5 Nguyen Van Toan, Nguyen Xuan Truong, Dao Ba Yen and Nguyen Thi Thu Cuc (2015), “Research results on the completion

of propagation procedure and new plantation of two rubber variety VNg 77-2 and VNg 77- 4 for the Northern mountainous

provinces”, Journal of Agriculture and Rural Development No 5

(2015), p 34-44

6 Le Quoc Doanh, Nguyen Van Toan, Nguyen Xuan Truong, Do Sy

An and Nguyen Huy Thuan, (2011) Decision No 1208 / BNN-TT on the recognition of the new agricultural plant variety, dated 6 June 2011 of the Ministry of Agriculture and Rural Development

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QD-1

INTRODUCTION 1.The need of this research

There are two most challenging factors for developing rubber in the Northern Mountainous Region (NMR) Firstly, low temperature conditions in winters (average minimum temperature from 10-150C) Secondly, soils use for rubber plantations are mostly steep, with the slope ranges from 150 to 200 In addition, the topsoil is frequently very thin, while the total nutrients and exchangeable cation capacity of rubber soils are poor and low These limitations need to be addressed

in order to sustainably develop rubber trees in the NMR One of the proposed solutions is to select suitable rubber varieties (good growth, high yield and cold tolerance), accompanying with breeding and cultivation techniques to develop rubber in the Northern mountainous areas Meanwhile, the research results on rubber trees in the NMR are limited in terms of quantity and research time, and to our knowledge, there has not been any systematic study to deal with such challenges Therefore, the study of the topic: "Research on technical measures to develop rubber trees in the northern mountainous provinces" is very necessary

2 The objects of the research

Successfully select cold-tolerant rubber lines and identify suitable breeding and cultivation techniques to meet the requirements

of sustainable rubber development in the Northern Mountainous Region

3 The subjects and scope of this research

3.1 Research subjects

The study conducted on 13 common rubber clones: IAN 873, RRIC 100, RRIC 121, GT1, RRIM 600, RRIM 712, RRIV 1, RRIV

106, RRIV 124, LT 74, VNg 77-2, VNg 77 -4, VN 73-46 and 28 newly created domestic rubber clones (detailed in the introduction of rubber varieties, appendix 2)

3.2 Research scope

- Assess the cold tolerance of clones planted in nursey gardens

at the establishment stages of aged 2 and 3 Materials for analyzing physiological and biochemical indicators including morphological and anatomical characteristics of leaves were sourced from 1- 2 whorl leaf polybag rubber seedlings in the nursery gardens and grafted branches in the breeding gardens

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2

- Research on rubber tree development is limited to selective breeding methods, breeding techniques, cultivation methods in the immature stages, aiming at increasing growth ability, latex yield and shorten the immature period At the same time, it aims to increase the capacity of supplying cold-tolerant rubber varieties to meet the demand of expanding rubber growing areas in the Northern mountainous provinces The research content of this thesis does not aim to develop rubber trees in a multi-purpose direction In addition,

in order to meet the research objectives, this study inherited the research experiments and results, which were already conducted and implemented during 2011-2015 by the author of this research These inheritances have been agreed and allowed by the Northern Mountainous Agriculture and Forestry Science Institute (NOMAFSI)

4 The scientific and practical significance of the research

Scientific significance

The thesis is a systematic study on the botanical characteristics

of rubber trees with a focus on finding the relationships between morphological, leaf anatomy and physiological and biochemical criteria in relation to low temperature tolerance This is the basis for the rapid selection of individuals oriented to the goal of selecting cold-tolerant rubber varieties, contributing to reducing the breeding time The research results of the thesis have provided the basis for completing the rubber tree breeding technique, which is the foundation for the continuation of research on rubber breeding techniques in the ecological conditions of the Northern mountainous areas; as well as plays as reference materials for educational purposes regarding rubber trees in the Northern mountainous areas

Through nutritional field experiments, the thesis has sugguested the hypotheses about the effect of fertilizer quantities on the changes of mineral nutrient contents in rubber leaves and some soil chemical characteristics These are good suggestions for further research

Practical significance

The study sucessfully selected cold-tolerant, high-yield rubber clones to recommend for rubber mass production in the NMR The study has completed the breeding process; supplementing some technical measures in rubber production in the NMR such as selecting suitable intercropped crops on immature rubber gardens and

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3 appropriate fertilizer quantity These are useful recommendations for provinces where rubber tree are planting, both for rubber farmers and staff involved in directing the rubber production

5 Novel contribution of the study

The dissertation is the firstly comprehensive research project

on newly created and imported rubber genetic resources tested in the NMR, selected some recommended rubber lines for production: DVT

27, RRIV 124 and VNg 77-4 In particular, VNg 77-4 has been recognized as cold tolerant rubber varieties and are allowing to use for mass production in the Northern moutanous region The desertation is also the first research in Vietnam to study cold tolerance of rubber varieties in combination with assessing morphological expression, leaf anatomical characteristics and changes of biochemical and biochemical indicators under the effects

of low temperature conditions; establishing a multivariate correlation function which reflects the relationship of damage index of some leaf

anatomy with physiological and biochemical criteria

The thesis has clarified the differences in the breeding season compared to the traditional region; techniques for planting and tending nursery gardens; the important role of potassium fertilizer in increasing cold tolerance for rubber in nursery stage The study also sucessfully recommended a technical process of propagating rubber trees for the NMR (recognized by the Ministry of Agriculture and Rural Development as a technical advance)

The research results of the thesis initially affirmed that: galangal (Alpinia officinarum) is an efficiently intercropping crop

during the period of from closed canopy to the early commercial stage of rubber tress; the 1.5 time increased amount of fertilizer compared to the technical procedure issued on 2012 (60 kg N + 60 kg K2O + 30 kg K2O / ha) resulted in the increased growth of rubber trees in the immature period, and significantly increased latex yield in the first exploited years; establishing a linear relationship between yield and rubber perimeter; suggesting initial assesments on leaf nutrient content and soil chemistry characteristics of fertilization treatments

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Chapter 1 LITERATURE REVIEW AND SCIENTIFIC BACKGROUND

OF THE RESEARCH

1 The scientific background of the research

Rubber trees do have requirements on climatic conditions such

as evenly distributed rainfall of over 2,000 mm / year without dry season, the number of rainy days is from 125-150 days /year; maximum temperature from 29-340C, minimum about 200C with average monthly temperature from 25-280C The main difference in climatic conditions in rubber plantations in traditional and non-traditional regions is the low average temperature It causes not only morphological damages on the visible parts of rubber trees: leaves, stems, roots but also directly affect the physiological processes in plants: photosynthesis, respiration and water exchange

Each rubber variety has different leaf morphological characteristics (varies with cultivated conditions and the environment) This feature is the difference between varieties and is used in the evaluation and identification of varieties at nursery and rubber plantations at immature stages

Rubber propagation by grafting is still the main breeding method, which is applied to produce leafy seedlings for new planting

in the world as well as in Vietnam The two important plant materials used are the scion cuttings taken from the breeding garden and the rootstock The compatibility of these two materials plays a critical role

in deciding the survival rate of rubber trees after transplantation Prolonging the standard rootstock will extend the seed production time Fertilizers have been shown to be able to improve growth ability, shorten growth duration in unfavorable growing areas, and contributing

to latex yield improvement Rubber trees need fertilizer mainly with macronutrients: N, P, K and partly Mg Potassium plays a role in increasing plant tolerance in low temperatures.In addition, unfavorable condition increases the demand for fertilizers for rubber trees

Appropriate intercropping on rubber plantations can bring economic benefits, improve plant growth, maintain and improve the physical and chemical characteristics of the soil Moreover, based on the characteristics of each type of crop and the growth stage of the rubber plantation to choose suitable intercropping crops

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2 Some comments are drawn from the literature review

Suitable variety is the most important factor for growing rubber

in unfavorable areas Evaluation of newly created and imported rubber clone lines is still the method of selecting rubber varieties being used in the world Some of the main criteria used in evaluating variety selection in non-traditional areas are: growth ability, latex yield and cold tolerance based on the method of Huang & Pan introduced in 1992 Aside, some physiological and biochemical indicators are also used to assess the cold tolerance of rubber trees such as relative water content, chlorophyll fluorescence, photosynthetic pigment (chlorophyll a and chlorophyll b, total chlorophyll and carotenoid), proline and catalase content Some of the criteria used in the anatomy of the leaves are: thickness of the main veins and lamella, the lamina layer and the chlorenchyma Cloning by grafting is still the main method used to create materials for new plantation

In order to sustainably develop rubber trees in the Northern region, the selection of suitable varieties and breeding techniques that suits natural and socio-economic conditions of the region plays an important role Currently, the research of variety sustability for the NMR is poorly implemented, especially in terms of research duration

to evaluate the performance of newly created domestic clones as well

as new imported varieties To date, there has not been any studies on cold tolerance of varieties regarding physiological, biochemical criteria and leaf defoliant characteristics In addition, solutions to deal with the effects of low temperature conditions to the breeding processes such as preparation of scion cuttings, planting time, seedling care have not yet been identified

In the NMR, studies on inorganic fertilizer application were only evaluated its effect during the first years of applications (1-2 years) and there are no outcomes of soil nutrient composition and rubber leaf evaluation A number of studies on intercropping have shown to

be effective when rubber trees are intercropped in the early stage in the region However, there has not been any studies on intercropping with rubber trees during the first plantation years when the tree canopy begins to close until it is fully closed

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Chapter 2 MATERIAL, CONTENT AND RESEARCH METHOD 2.1 Research material

- Rubber variety:

- Rubber varieties: IAN 873, RRIC 100, RRIC 121, GT 1, RRIM 600, RRIM 712, RRIV 1, RRIV 106, RRIV 124, VNg 77-4, VNg 77-2, LT 74 and VN 73-46

- Clone lines: 28 new domestic hybrid clones provided by the Vietnam Rubber Research Institute

- Fertilizers: Nitrogen Ha Bac (46% N), Lam Thao phosphate

(Supephosphate 16% P2O5), Kaliclorua (60% K2O), Maggie phosphate (16% MgO)

- Intercropping crops: Mulato (Brachiaria mulato), Brizantha

(Brachiaria brizantha), Galangal (Alpinia officinarum), Turmeric N8 (Curcuma longa) Mondograss (Ophiopogon japonicus Wall.)

- Other materials: Seed rubber tree, grafted rubber tree

branches (4-5 whorl leaves), grafted buds, 1- 2 whorl leaf polybag rubber seedlings

2.2.Time and research location

2.2.1 Research time: All the research components were undertaken

from 2011- 2019

2.2.2: Research locations: NOMAFSI; Chieng Ban and Chieng

Kheo communes, Mai Son district, Son La province; Khong Lao commune, Phong Tho district, Lai Chau province; and Minh Quan commune, Tran Yen district, Yen Bai province

2.3 Research content

Content 1: Study on agro-biological characteristics and cold tolerance of some rubber clones in the NMR;

Content 2: Research on rubber breeding techniques;

Content 3: Research on some cultivation techniques for rubber trees

2.4 Research method

2.4.1 Research method of content 1: Study on agro-biological characteristics and cold tolerance of some rubber clones in the NMR

2.4.1.1 Experiment design

Experiment 1: Research cold tolerance in the field

In the variety comparison experiment, assess the level of damage to the entire number of plants in the treatment On the nursery and the plantation demonstration, randomly evaluate the level

of morphological damage of rubber morphology with 5 replicates (each replicate with 2 rubber rows, 10 trees in each row)

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Experiment 2: Research on growth ability and latex yield of

some rubber clones

The experiement consists of 7 treatments, each treatment is a clone Each treatment includes 5 plants with three replicates and was completely randomized block designed, which ended up with a total

15 plants Design density of 571 trees / ha (spacing of 7 x 2.5 m) Plants used for the experiment were the polybag rubber seedlings with 2 stabe whorl leaves Experiemental clones were including DVT

27, DVT 30, DVT 54, RRIV 124, VNg 77-2, VNg 77-4 and LT 74

Experiement 3: Study on morphological and anatomical

characteristics of leaves of some rubber varieties

The varieties selected for research include: IAN 873, RRIC

121, RRIV 1, RRIV 124 and VNg 77-4 Varieties on the nusery fields were uniformly cut in February The timing to select and eveluate morphological criteria was when the grafted branches had 5 stable whorl leaves The leaflets selected for morphological evaluation were typical leaves in the middle of the selected leaf layers Leaf samples were also taken for anatomical analysis The experiment was arranged in completely randomized blocks of 10 plants / variety x 3 replicates

Experiement 4: Research some physiological and biochemical

criteria in rubber leaves

Experiement 4a Assess the relative water content of some

rubber clones

Five varieties were selected for the research including: IAN

873, RRIC 121, RRIV 1, RRIV 124 and VNg 77-4 Two factors experiment were arranged in completely randomized block with 5 rubber varieties, 2 thresholds for low temperature processing time of

80C for 60 hours, 120 hours and 3 replicates Number of experimental leaves: 8 leaves / variety / replicate Experimental leaves were collected at 40 days of age The sample preparation and determination of relative water content processes used the method of Balsamo, RA (2006) (Appendix 16)

Experiement 4b Study the response of some biochemical and

biochemical indicators of some rubber clones in low temperature conditions

The studied rubber varieties including: IAN 873, RRIC 121, RRIV 106, RRIV 124 and VNg 77-4 Each variety is a treatment, each treatment consists of 3 plants (2 whorl leaf) placed under 80C for 60 hours Methods of Sarkar.J (2013) was used for sample

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8 preparation and low temperature treatment (Appendix 17) The experiment was arranged in completely random blocks

2.4.1.2 Parameters and observation methods

Climatic condtions: Collecting secondary data (monthly average temperature, rainfall and number of sunshine hours) at some weather stations in rubber growing provinces: Lai Chau, Son La, Phu Tho and Yen Bai in January 2011; collect secondary data (detailed daily values of mean temperature, maximum temperature, minimum temperature) from January to March in Phu Ho commune, Phu Tho town, Phu Tho province and Chieng Ban commune, Mai Son district, Son La province

Assessing cold tolerance through morphological expression: Assessing the level of damage and recovery after cold stress of rubber trees based on the evaluation method developed by Huang & Pan (1992) and adjusted by Vu Van Truong (2009)

The criteria for leaf morphological assessment are described based on the guidelines for the evaluation of the distinctness, uniformity and stability of rubber varieties of the International Union for the Protection of New Varieties of Plants (UPOV) and the identification rubber variety handbook isued by the Vietnam Rubber

Research Institute in 2012 Westen The anatomy of leaves by the

method described by Tran Cong Khanh (1979)

Biochemical and physiological criteria:

+ Photosynthetic pigments were separated by 80% acetone solution (Westen, 1984), the adsorption spectra of the extracts were measured at the wavelengths of 663.2 nm, 646.8 nm and 470 nm by the adsorption spectrophotometer UV-VIS GENESYS 10uv (Thermo Electron Corporation, USA) to determine leaf tissue content by the method described by Nguyen Van Ma et al (2013)

+ Chlorophyll fluorescence was measured with OS30p + (OPTI-SCIENES, USA)

+ Proline content was determined by optical method based on the method described by Nguyen Van Ma et al (2013)

+ The enzyme catalase activity was determined by the titration method described by Nguyen Van Ma et al

+ Relative water content was determined by the method of Balsamo, RA (2006)

Agronomic parameters: The growth indicators (stem perimeter

and primary bark thickness) were evaluated based on the tecnhnical standard 10 TCN 9002: 2006 (Rubber - agronomic indicators used in

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9 research Latex yield: gram/tree/cut : latex was frozed at the latex collection bowls using acetic acid solution 3% and weighed after drying by hanging in the dried locations, without directly contacting the sunshine for at least 3 weeks

2.4.2 Research method for contents 2: Research on rubber breeding techniques

2.4.2.1 Experiment design

Experiement 5: Study Huang & Pan on some technical

measures on nusery

Experiement 5a Effect of planting density on growth capability

and grafting cuting productivity of rubber plants in the nusery

The experiemnt includes three densities, each is a treatment with three replicates with 150 m2 /replicate The experiemnt treatment (T) included:

T1: Density of 25.000 trees/ha (1 x 0,4m, control)

CT1: 20 tons manure + 1 ton P2O5 + 500 kg lime/ha (control) CT2: 25 tons manure + 1 ton P2O5 + 500 kg lime/ha

CT3: 30 tons manure + 1 ton P2O5 + 500 kg lime/ha

CT4: 35 tons manure + 1 ton P2O5 + 500 kg lime/ha

Experiement 5c Determination of suitabe pruning time

The experiment consists of four pruning season, each season

is a treatment that was designed in a randomized complete block with

3 replicates; each consists of 30 rubber trees Experimental treatments were:

T1: Pruning in September

T2: Pruning in October

T3: Pruning in November T4: Pruning in February

Experiment 6: Determination of suitable grafting season in

the nursery

The experiment consists of 5 grafting seasons, each is a treatment which was designed in a randomized complete block with 3 replicates, each consists of 30 rubber trees

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10 The treatments were:

T1: Grafting in June

T2: Grafting in July

T3: Grafting in August

T4: Grafting in September T5: Grafting in October

Experiment 7: Effects of potassium fertilization quantity on

some biochemical and biochemical indicators of rubber seedlings in low temperature conditions

The experiment was designed in randomized complete blocks, in which each variety was seperately arranged The experiment included 5 treatments (changing the concentration of K2O) Each treatment had 10 plants with 3 replicates The total number of experimental plants was 150 trees /variety After being fertilizered with amount of 1 gN + 1 g P2O5/seedling, the polybag rubber seedlings then were applied with potassium fertilizer by the amount of 0,00, 0,25, 0,50, 0,75, 1,00 g K2O/ each seddling, depending on the treatments Rubber trees were fertilized at 35 days after grafting, and 20 days after fertilizing, the plants were measured for growth and heat stress treatment at 8°C (12h for RRIV 124 and

48 hours for VNg 77-4) using the method of Sarkar.J (2013) (Appendix 17)

2.4.2.2 Parameters and observation methods

Agronomic parameters

Diameter of grafted buds, diameter of grafted branches, number of whorl leaf, grafting survival rate were evaluated based on the technical standard 10 TCN 9002: 2006 Some other criteria: Number of horizontal buds (buds), height of grafted branches (cm), sufficient rate of woody graft (%), number of sufficient grafted cuttings / 1m of grafted branches (cuttings), sufficient rate of grafted branches (%), total meters of branches / ha (m), height of grafted buds (cm), leaf size, successful seedling rate (%), propagation coefficient (ha / year)

Biochemical and physiological criteria

Photosynthesis pigments, chlorophyll fluorescence (Fv / Fm), proline content (µg / g of fresh leaves), catalase enzyme activity (U /

g of fresh leaves) were conducted using the method described in the subheading 2.4.1.2

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