單 妍

(昆明醫(yī)科大學(xué)海源學(xué)院 昆明 651700)

主要經(jīng)濟(jì)竹種的微體快繁及苗木培育技術(shù)研究

單 妍

(昆明醫(yī)科大學(xué)海源學(xué)院 昆明 651700)

選擇35個經(jīng)濟(jì)竹種采用以芽繁芽的方式進(jìn)行微體快速繁殖試驗及苗木培育試驗。結(jié)果顯示，以種子作為外植體組培效果最好，其他依次為幼年竹節(jié)芽、小枝芽、大枝芽，成年竹節(jié)芽組培效果最差。相同煉苗條件下，毛環(huán)刺竹、實心瓜多竹、孝順竹、黃金碧玉竹、非洲銳藥竹、麻竹和大佛肚竹更易存活和發(fā)筍。苗木培育試驗結(jié)果顯示，蛭石是較為理想的煉苗基質(zhì)，腐殖土及“30%鋸末＋70%紅土”均為較好的育苗基質(zhì)；組培苗移栽后施以N、P、K復(fù)合肥作為葉面肥，配以微量元素的追肥，可提高組培苗的成活率。竹子組培苗便于運輸且成本低，因此成為生產(chǎn)竹苗的最佳選擇。

經(jīng)濟(jì)竹種；組織培養(yǎng)；苗木培育

In the past years，Yunnan has been geared up by the afforestation projects such as the natural forest protection project，public welfare forest construction，shelter forest construction，and returning farmland to forest，which has generated around 2 million ha of fastgrowing and high-yield plantation till 2016.Bamboo has been the main plant and played a significant role in forest coverage in Yunnan province.Therefore，it's been a hotspot to figure out how to cultivate high-quality bamboo plantation in large area.

Bamboo is characterized by its long flowering cycle with unpredictability and infertility，which makes regular sexual cultivation very difficult.Therefore，bamboo is mainly relied on asexual propagation such as burying rhizomes，buryingculmsandburyingnodesetc. Requiringlargeamountofmaterial，highcostof transportation and intensive labor but still ending up with low propagation coefficient，such asexual propagationways cannot meet with the growing demand for bamboo of industrial utilization.Compared to the traditional asexual propagation，tissue culture has the advantages of being high coefficient，rapid，easy to rejuvenate from disease. According to researches，one bud of a clumper can propagate 10 000 plants within 1 year by tissue culture，but only 5～10 plants by burying nodes[1].The most possible way to realize bamboo micropropagation is to induce buds from embryo，young or mature buds，and apical meristem growing on appropriate medium without callus as intermediary，and then multiplicate shoots from buds in tubes.This way of induction is efficient，easy and low cost，which makes it possible for industrializing nursery[2].

1 Experimental site

Yunnan Bamboo Nursery and Songming Songyang Nursery.

2 Experimental materials and methods

2.1 Materials

The bamboo species and the explants used in this study are listed in Tab.1.

Tab.1 The explants of different bamboo species and material

The medium，growth regulators and the chemical reagents used in this study are as follows:

1)Nutrients at culture stage:MS medium，coconut milk.

2)Growth regulator:BA，IBA，KT，NAA.

3)Chemical reagents:2.5%sodium hypochlorite，potassium permanganate，Twain(as surfactant)，0.1% mercuric chloride，75%ethanol，potassium dihydrogen phosphate，carbamide，carbendazol，microelements，compound of N，P，K.

2.2 Experimental methods

2.2.1 Selection of explant

Selection of explant is the key point for bamboo tissue culture.If seeds selected，they should be plump and fresh seeds with distinctive color and no insects.If nodes selected，they should be nodes from lignified branches，with short diameter but no wounds or bare buds.On the premise that the explant's viability is ensured，the explant shall be as short as possible so as to reduce browning and contamination.

2.2.2 Sterilization of explant

No matter what explant is used，it should be thoroughly sterilized and disinfected before inoculation so as to prevent the surface or inside of the structure from being infected by microorganism.

1)Sterilization of seeds:Remove husk ofthe chosen seeds and then soak in 2.5%sodium hypochlorite(with 4 drops of Twain)for 15 min，wash seeds with sterile water，soak the seeds in sterile water(35℃)for 30 min，followed by soaking the seeds in 30 ppm potassium permanganate solution for 10 hours，wash the seeds with sterile water.Move to the sterile console，sterilize the seeds with 0.1%mercuric chloride(with 4 drops of twain)，wash with sterile water 5 times，about 5 min per time[3].

2)Sterilization of branch bud and node bud:Soak the chosen branch buds or node buds in sterile water for 2～3 hours，then in 75%ethanol solution for 1 min； wash with sterile water，put the material into refrigerator (10 centigrade)for 1 d，scrub the material twice with 75%ethanol solution，then soak the material in 2.5% sodium hypochlorite(with 4 drops of Twain)for 15 min，wash with sterile water 3 times.Move to the sterile console，soak the material in 0.2%mercuric chloride (with 4 drops of twain)for 15 min，wash with sterile water 5 times，5 min per time[3].

2.2.3 Culture medium

MS medium is the most one frequently used as basic medium for bamboo tissue culture.Certain adjustment is made at different stages in favor of best growth to bamboo.Macroelementsisinaccordancewiththe standard MS，1/1，1/2 and 3/4.Microelements，iron salt and cane sugar remain unchanged；Organic nutrients like vitaminisadjustedaccordingtoB5medium (Gamborg，1968).Growth regulators such as BA，IBA，KT and NAA etc with proper concentration；pH value 5.8～6.2；agar 0.65%.

2.2.4 Culture condition

Temperature:25～28℃，illumination:12h/d，1 600 Lx

2.2.5 Hardening plantlets and transplantation

Expose in-vitro plantlets under natural light for 5～7 days without cover when roots of plants from seeds grow to 2～7 cm long，more time required for mature bamboo plantlets.Then wash the in-vitro plantlets with tap water and soak in carbendazim solution for 10 min. Put plastic film and shade，djust temperature，humidity and sunlight properly.Removethefilm whenthe plantlets generate new leaves or shoots.Vermiculite and perlite are used as growth medium for comparison.

3 Results and analyses

3.1 Difficulty levels of different bamboo seed types

Bamboo seeds from 10 species were chosen for the experiment.One sterilized seed was inoculated in each tube with MS medium(supplemented with BA 0.2 mg/L).The seeds were sprouting since the 4thday.The germination rate was recorded.When the germs grow to 1 cm long，they were transplanted to new medium of MS (m)＋BA 3.0 mg/L.The contamination rate was recorded.When node buds begin to tiller to form multiple buds，the tiller rate was recorded.The multiple buds were divided repeatedly for subculture in new medium(3/4 MS(m)＋BA 2.0 mg/L＋KT 0.5 mg/L＋coconut cream 100 mL/L[4]).The multiple buds were inoculated in rooting medium(1/3 MS(m) ＋NAA 2.5 mg/L＋I(xiàn)BA 2.5 mg/L[5]).Plantlets grow roots within 10 days.The rooting rate and seedling rate were recorded.Comparison analysis was conducted for the 5 different rates of 10 bamboo species as below in Tab.2.

Tab.2 Tissue culture results of 4 types of seeds from 10 bamboo species

According to Tab.2，all tested bamboo seeds succeeded in seedling，among which Melocanna baccifera，D.membranaceusvar.grandis，D.barbatus，and Thyrsostachys siamensis reached 100%of rooting rate.

1)Germination rate of M.baccifera is the highest one(96.5%)，while D.brandisii has the lowest germination rate(81.6%).The reason for this is that the seed of M.baccifera is pome with volume of over 7 cm long and 5 cm diameter，which can provide enough nutrients for germination.The 4 bamboo species with caryopsis seeds get 2ndhighest germination rate，because the pericarp is thin and closely with seed coat，which makes germination easier.The nut seeds of 2 bamboo species，covering hard pericarp over 1 mm thick and separate from seed coat，had diameter over 1 cm which can providenutrientsforgermination.AsforD. brandisii，D.latiflorus and D.fuminensis，whose seeds are nut-caryopsis，the nutrients provided by the lower caryopsis part is relatively less and it's hard for the upper nut part to absorb water，which resulted in a lowest germination rate.

2)Contamination rate:M.baccifera is the highest one(74.6%)，while Th.siamensis the lowest one (8.7%).This is because M.baccifera is the biggest sized seeds with large surface exposed to contamination，whileTh.siamensis has relativelysmaller exposed surface.

3)There is no obvious difference regarding tiller rate，rooting rate and seedling rate among different bamboo species.

3.2 Difficulty levels of different bamboo species

Thirty sterilized branch nodes of 8 bamboo specieswere selected and chipped off two ends of node buds in clean bench.Keep one bud，and The node about 1 cm long on two ends kept on bud was inoculated in the medium(3/4MS(m)＋BA 4.0 mg/L＋KT 0.5 mg/L[3])for bud induction.The same medium as for bamboo seeds was applied for multiplication and rooting. Comparison analysis was conducted for the 4 different rates of 8 bamboo species as below in Tab.3.

Tab.3 Culture results of branch nodes from 8 species

The result shows that all tested 8 bamboo species can be cultured to rooted plantlets，among which D. latiflorus performed best while Neosinocalamus distegia worst.The total performance from the best to the worst was as follows:D.latiflorus＞D.fuminensis＞Chimonocalamusdelicatus＞D.brandisii＞D. farinosus＞Fargesia songmingensis＞D.minor＞Neosinocalamus distegia.

3.3 Difficulty levels of different explants

Seeds，branch buds，primary branch buds，young node buds and mature node buds of D.latiflorus were selectedasexplantsformultiplebudsinduction，proliferation and rooting.The results were as follows in Tab.4.

Tab.4 Culture results of different explants of D.latiflorus

Tab.4 showed that among all the explants of D. latiflorus，the best culture result came from seed while the worst from mature node bud.Total performance from the best to the worst was seed＞young node bud＞branch bud＞primary branch bud＞mature node bud. Young node bud and branch bud performed better than primary branch bud and mature node bud，because juvenile tissue had a higher morphogenetic capacity than mature tissue.

3.4 Growthofplantletsexercisedin different substrates

The experiment was taken in green house coveredwith plastic film and shade，using vermiculite and perlite as substrate for comparison.In the first week，water spray was applied 6 times during 11∶00 a.m.－16∶00 p.m.everydaytokeephumidityofthesmall environment.Sincethe2ndweek，solutionof monopotassium phosphate，Urea and carbendazim was sprayed on the plantlets every 3 days.Meanwhile，keep the substrate water content.The survival rate and shooting root of plants were counted after 45 days as in Tab.5.

Tab.5 Growth in different substrates of 5 bamboo species

The result indicated that all tested bamboo species could survive and shoot after transplant(Tab.5). Almost all tested bamboo species performed better in vermiculite than in perlite.The selected vermiculite，in theshapeofpowder，hadadvantagesofgood permeability，highabsorption，smallchangeon temperature and moisture，which could facilitate growth and tiller of plantlets and enable fertilizer to release to the substrate slowly.The minerals in the substrate itself could provide nutrients for the plantlets at the primary growth stage.The selected perlite，in shape of particle，waslightandventilativebutshortofnutritional ingredients and could not hold moisture well.

3.5 Survivalofplantletsexercisedunder different conditions

Bambusa ventricosa plantlets were transplanted in vermiculite and perlite and placed in thermostat and green house respectively.1)Prepare 15 seedling bags with vermiculite and perlite respectively，one plantlet in one bag.Spray enough water and cover the bags with transparent plasticbags.Putallthebagsintoa thermostat and fix inner temperature at 27℃.Place the thermostat over against a window for light.It showed the plantlets in thermostat died of etiolation and withering in succession within 12 days.Comparatively the plantlets in vermiculite grew better and survived longer than those in perlite.2)Transplant the plantlets into green house with vermiculite and perlite respectively as substrate.Water periodically andkeeprecordsofplantletsgrowth. Plantlets in green house performed well，with a survival rate of 85%and shooting rate of 90%.It came to the sameconclusionthattheplantletsinvermiculite performed better than those in perlite，with a higher survival rate and shooting rate.

The thermostat can keep the required temperature and the plastic bags can hold required moisture for the substrate.But the restrictednatural light intothe thermostat limited photosynthesis of the plantlets.The green house was facilitated with adjustable temperature，moisture and sunlight，which enabled the plantlets to survive and perform well.

3.6 Survival and growth of different bamboo species after being exercised

Plantlets of 14 bamboo species were transplanted intogreenhousewithvermiculiteandperlite respectively，amongwhich 5speciesincludingB.mutiplex were transplanted in vermiculite while the others including D.hamiltonii in perlite.The records of survival rate and shooting rate after 45 days were as below in Tab.6.

Tab.6 Survival and growth of 14 bamboo species

Tab.6 showed that 4 out of 5 species in vermiculite came up with a survival rate of 100%.B.multiplex，B. vulgaris cv.vittata and Oxythenathera abysinica reached 100%for both survival and shooting rate.Five out of 9 species in perlite reached 100%for survival rate，and 3 out of 9 reached 100%for shooting rate.Only D. latiflorus and B.ventricosa reached 100%for both survival and shooting rate.

3.7 Growth of plantlets in different substrates

Theplantlets of D.latiflorus were transplanted into 5 different substrates including black soil，red soil，30% saw dust＋70%red oil，humus and fine sand.The results were listed as below in Tab.7.

Tab.7 Growth of D.latiflorus(one-year-old)in different substrates

Tab.7 showed that the plantlets of D.latiflorus could survive and shoot in all tested substrates，which performed the best in vegetable earth and the worst in fine sand，those in 30%saw dust＋70%red oil，red soil and black soil lied between the best and the worst. The humus was a kind of organic matter buried in theunderground，which was decomposed into incomplete organic matter by long-term decay fermentation under hypoxic conditions.The soil particle size was uniform and moderate，and the permeability and the capacity of water and fertilizer were good，which was suitable for root growth.Saw dust was light and held water and fertilizerwellinsteadofnutrition.Redsoilwas characterizedbyhighmoisture，lowdensity，high intensity and low compressibility with certain minerals. Mixture of saw dust and red soil at a certain proportion might be excellent propagation substrate.Black soil contained an average of 3%～10%organics，which had good expansion coefficient and water capacity，but poor effectiveness.Fine sand had the smallest particles with a certain water holding capacity instead of nutrition.

3.8 Growth of plantlets under different managementsDifferent nursing methods were applied tothe plantlets of D.latiflorus，the results were as below in Tab.8.

Tab.8 Growth of D.latiflorus under different managements

The results showed that D.latiflorus performed comparatively well when N，P，K compound was sprayed toleaves.Whenmicro-elementswereaddedto substrate，the plantlets performed better.The plantlets without apical dominance were better than those with apical dominance.The plantlets only watered performed poorly.Urea was an organic compound with C，H，O and H.N，P，K compound was very helpful，because P was very important to the growth of leaves and roots at primary stage and K helped to promote the growth of leaf vein.Meanwhile micro-elements were very important too.Capping of the plantlets could get rid of apical dominance which led to more shooting.

3.9 Quality of plantlets from different propagation methods

Analysis was made on plantlets from seeds，cuttings，node burying and tissue culture，the results were as below in Tab.9.

Tab.9 Quality of D.latiflorus(one-year-old potted plantlets)from different propagation methods

The results showed that the plantlets from seeds and tissue culture of D.latiflorus had more culms per bag，whichwerelightinweight，comparativelysmaller diameter and height，and lower cost.These plantlets were easy for commercial application.

4 Conclusions

1)When seeds are chosen as explant，stricter sterilization shall be taken to reduce contamination，especially for large-sized pome seeds.For the thickpericarp and hard nut seeds，more time shall be given to soak the seeds to make germination easier.

2)From Table 2，3 and 4，we can see that seed is the best explant for tissue culture，followed by young node bud and branch bud.However，due to rarity of bamboo seeds，dynamic organization close to base culm is recommended for explant，such as young node bud and branch bud.

3)Vermiculite is good substrate for tissue culture plantlets.For one-year-old plantlets of D.latiflorus，humus is a good substrate，followed by mixture of saw dust(30%)and red soil(70%).

4)Among all the tested bamboo species under the same condition，B.balcooa，Guadua amplexifolia，B. multiplex，B.vulgariscv.vittata，Oxythenathera abysinica，D.latiflorus and B.ventricosa are easier to survive and shoot，while Phyllostachys aurea is the most difficult one.

5)Management is very important.For the plantlets of D.latiflorus，NPK compound as leaf fertilizer and micro-elements as additional fertilizer can enhance the survival rate and promote the plantlet growth.Capping of bamboo plants can promote more new shoots.

6)Plantlets from seeds and tissue culture are more cost-effective and easier for transportation than those from cutting and node burying.However，bamboo seeds are too rare to be obtained.Therefore，tissue culture is the best propagation method for commercial production.

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Study on Techniques of Tissue Culture and Propagation for Thirty-five Economic Bamboo Species

Shan Yan
(Kunming Medical University Haiyuan College，Kunming 651700，Yunnan，China)

Thirty-five economic bamboo species were selected for the experiments of micropropagation.Among all the explants，seed is the best one，followed by young node bud，small branch bud and big branch bud，while mature node bud is the worst.Under the same conditions，Bambusa balcooa，Guadua amplexifolia，B.multiplex，B.vulgaris cv. vittata，Oxythenathera abysinica，Dendrocalamus latiflorus and B.ventricosa are relatively easier to survive and shoot，while Phyllostachys aurea is the hardest one.Vermiculite is the most productive growth substrate.While for one-year tissue culture plantlets of D.latiflorus，vegetable soil is an excellent substrate，followed by the mixture of saw dust (30%)and red clay(70%).Foliar fertilizer of N，P，K compound combined with micro elements on foliage can increase the survival rate of plantlets.Capping of bamboo plantlets can boost shooting.Bamboo plantlets from seeds and tissue culture are both easy to transport and low cost.Moreover，tissue culture plantlets become the best choice due to the rarity of bamboo seeds.

economic bamboo，tissue culture，micropropagation，bamboo cultivation

10.13640/j.cnki.wbr.2017.01.004

意大利ONLYMOSO公司資助項目(編號:2014003)；云南珍竹農(nóng)業(yè)科技有限公司資助項目(編號:2011006)。

單妍(1981－)，女，在讀碩士，講師，研究方向為生物工程。E－mail:41513075＠qq.com。