Banana protocol

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Weaning (acclimatization) of in vitro-produced banana plants.

Abstract –– Introduction. The protocol describes a method for promoting the acclimatizationand protection of rooted in vitro banana plants between rooting in the laboratory and their esta-blishment in a nursery. The principle, key advantages, starting plant material and time requi-red are presented. Materials and methods. This part describes the weaning houseinstallation, the weaning medium used to transplant the vitroplants, the two steps for acclimatizingthe plantlets: receipt of rooted laboratory plants in flasks and planting, and factors affecting thegrowth of the plantlets in weaning conditions (light, misting system, temperature, humidity,wind, pest control and nutrition). Possible problems for troubleshooting are listed. Results.Under normal conditions, new roots appear 4–5 days after the start of weaning, and new leavesstart to grow within 8–10 days. At the end of the weaning phase, young plants are healthy, welldeveloped and they can be established in a nursery.

South Africa / Spain / Musa sp. / methods / vitroplants / weaning

Sevrage (acclimatation) de vitroplants de bananier.

Résumé –– Introduction. Le protocole décrit une méthode qui favorise l'acclimatation et laprotection des vitroplants racinés de bananier entre la phase d’enracinement en laboratoire etleur établissement en pépinière. Le principe, les principaux avantages de la méthode, le maté-riel végétal de départ et le temps requis sont présentés. Matériel et méthodes. Cette partiedécrit l'installation de la structure de sevrage, le milieu de sevrage utilisé pour transplanter lesvitroplants, ainsi que les deux étapes nécessaires pour acclimater les jeunes plants: réceptiondes plantes de laboratoire racinées en flacons et leur plantation, et facteurs affectant la crois-sance des jeunes plants en phase de sevrage (lumière, brumisation, température, humidité,vent, contrôle des parasites et nutrition). Des problèmes éventuels sont énumérés. Résultats.En conditions normales, les nouvelles racines apparaissent en 4 à 5 jours après le début dusevrage, et les nouvelles feuilles commencent à se développer en 8 à 10 jours. À la fin de laphase de sevrage, les jeunes plantes sont saines, bien développées et elles peuvent être trans-férées en pépinière.

Afrique du Sud / Espagne / Musa sp. / méthode / vitroplant / sevrage

1. Introduction

Application

This protocol aims at promoting the accli-matization and protection of rooted in vitrobanana plants between rooting in the lab-oratory and their establishment in a nursery.

Principle

The key to successful weaning is to keepyoung, sensitive plants free from any formof environmental stress for the entireperiod. Therefore, the ideal weaning systemcan be regulated to control the most impor-tant environmental factors (light, water,temperature, humidity and wind).

1 Du Roi Lab., P.O. Box 1147, Letsitele 0885, South Africa, duroilab@mpu.co.za

2 Inst. Canario Investig. Agrar. (ICIA), Apartado Correos 60, 38208 La Laguna, Tenerife, Canary Islands, Spain

vgalan@icia.es

Weaning (acclimatization) of in vitro-produced banana plantsJohn Charles ROBINSON1, Victor GALÁN SÁUCO2*

* Correspondence and reprints

Fruits, 2009, vol. 64, p. 325–332© 2009 Cirad/EDP SciencesAll rights reservedDOI: 10.1051/fruits:2009026www.fruits-journal.org

Article published by EDP Sciences and available at http://www.fruits-journal.org or http://dx.doi.org/10.1051/fruits:2009026

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Key advantages

The key advantage of this weaning protocolis that the process allows for the protectionand stabilization of young rooted plantletscoming out of a sterile laboratory environ-ment, until they are large and hardy enoughto be transferred to the harsher environmentof a nursery shade house.

Starting material

This prototol uses rooted in vitro plants inflasks. The material required comprises verytender plantlets which are actually removedfrom their protective laboratory flasks insidethe weaning house. The plants are typically(2 to 5) cm tall, with brittle white stems,three to five soft green leaves, and severallong white primary roots growing from the

base of the stem. The plants are packedclose together inside the flask, but are sep-arated carefully from their agar gel-rootingmedium to start the weaning protocol(figure 1).

Time required

In an ideal weaning system, five to eightweeks are necessary from the receipt ofrooted plants in the weaning house to hav-ing them ready for sending to the hardeningnurseries. Under warm tropical conditions,a (5 to 8) cm plant will be produced in fiveto six weeks, whereas under cooler sub-tropical conditions seven to eight weeks arerequired (figure 2).

2. Materials and methods

The weaning house

The weaning house can be a normal glass-house, or else constructed with a semi-cir-cular metal frame covered with clear poly-ethylene sheeting and with a double-doorentrance for hygiene. Inside, the traysshould rest on tables raised to workingheight, and an automatic misting system aswell as shade netting should be installed.Under extremely hot external conditions(above 35 °C), it is preferable to have a wetwall at one end, and extractor fans at theother end, to draw cool air through and pre-vent overheating. Alternatively, adequateventilation has to be provided. Under coolexternal conditions (below 18 °C), a heatingsystem should be installed to blow warm airthrough (figure 3).

The weaning medium

The common medium used in weaning traysis peat moss or sphagnum moss, but othertypes of milled organic material can also beused, e.g., coconut fiber (coir) (figure 4).These can also be mixed in various propor-tions with inert additives such as perlite, ver-miculite or styrofoam (e.g., 70% peat moss,30% vermiculite and 1 kg·m–3 dolomiticlime to adjust pH, if necessary). The mediumhas to be sterilized or pasteurized before use

Figure 1.Tender new banana vitroplants, just removed from the laboratory flask.

Figure 2.Newly-transplanted banana vitroplants (right) and nearing completion of the weaning period (left).

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since in vitro plants are highly vulnerable topathogen infection at this stage, particularlyby Pythium or Phytophthora spp. [1].

Use a weaning medium with an air-filledporosity (AFP) as close as possible to 20%,which is considered optimal. The AFP of amedium should not be below 10% andshould not be more than 25%. If AFP ishigher than 20%, then the dry-out rate isfaster and irrigations have to be more fre-quent. If the AFP is lower than 20%, the dry-out rate is slower and irrigations can be lessfrequent. If the AFP is less than 10%, thereis a high risk of compaction, water loggingand inhibited root growth.

Note: AFP is determined in the laboratory bysaturating a sample of the weaning mediumwith water and draining it, then measuringthe volume of water that drains out. Themedium has to be free-draining. The vol-ume of water draining out corresponds tothe volume of the air pores (AFP), which isexpressed as a percentage of the volume ofthe medium left after draining. A detailedprotocol for measuring AFP can be found athttp://www.geocities. com/RainForest/Can-opy/8771/air_porosity. htm.

Keep water-holding capacity (WHC) of theweaning medium between 40% and 50%.

Note: WHC is determined in the laboratoryby weighing a sample of weaning mediumwet after free-draining, followed by dryingand weighing the medium dry. The mass ofwater as a percentage of the wet mediummass is the WHC. The lower the AFP thehigher the WHC and vice versa. A detailedprotocol for measuring WHC can be foundat http://www.geocities.com/CapeCanaveral/Hall/1410/lab-Soil-06.html.

Keep the pH of the weaning mediumbetween 5.5 and 6.5. If lower than this,buffer with dolomitic lime.

Note: an easy protocol for measuring soil pHcan be found at http://www. geocities. com/CapeCanaveral/Hall/1410/lab-Soil-04.html.

Fertilize plants during weaning by one oftwo options: (A) Pre-mix the weaningmedium with a slow-release fertilizer (SRF)containing a NPK ratio of [2:1:2]. An accept-able mix would be 3–5 kg SRF·m–3 ofmedium (3–5 g·L–1), depending on the

nutrient release period [usually (70 to100) days] and the intended duration ofweaning. Any surplus release period willbenefit the young transplants in the nursery;(B) A soluble fertilizer application system(fertigation) can be installed, but do notexceed 60 mg·L–1 nitrogen in the appliedsolution. Applications are usually madeweekly. Such a fertigation program shouldbe recommended for the particular circum-stances by a competent nutrition expert.

Note: inoculation with arbuscular mycor-rhizae may be beneficial for the plantby increasing its growth rate, facilitatingnutrient uptake and increasing disease tol-erance [2].

Figure 3.Inside a plastic weaning house, showing trays with transplanted banana vitroplants on moveable tables, shade cloth protection, and overhead misting system.

Figure 4.Coconut coir weaning medium showing good air-filled porosity and excellent banana rooting potential, but causing salinity damage on lower leaf.

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The usual weaning tray is made of 32 densitymolded polystyrene and contains round orsquare tapered holes or plugs (30 mmdiameter × 60 mm deep) which have adrainage hole at the bottom. Variations inplug size and shape are used according topreference.

Note: there should be approximately 100 to200 holes per tray and 40 cm3 per hole, giv-ing a density in weaning of about550 plants·m–2 [3]. Spray plugs in polysty-rene trays before use, using an approvedsealant such as ‘Styroseal’ to prevent rootsfrom penetrating the walls of the tray, whichwould later make plant extraction difficult.Alternatively, use plastic trays which are

more hygienic, easier to clean and alloweasier extraction of plugs (figure 5).

Acclimatization of the plantlets

• Step 1. Receipt of rooted laboratory plantsin flasks and planting

Under humid conditions inside the weaninghouse, take off the lid and remove the clumpof agar-gelled plants gently from inside theflask, and separate them. Place individualplants in a bucket of clean water to washoff agar. To assist in planting, trim lengthyroots back to 2 cm. Alternatively, remove allroots neatly before planting [4] (figure 6).

Grade the plants into three sizes (i.e., small,medium and large), relative to the averagesize of plants received. Discard the very smallplants up to about 5% of the total plantsreceived. Also, discard any abnormal-look-ing plants and/or obvious mutations.

Note: careful size grading at this stage per-mits uniform growth in weaning trays andmakes grading at the nursery stage mucheasier (figure 7).

Dip the whole plant in a fungicide mixturebefore planting in trays. Captab or a locallyused fungicide can be used at recom-mended concentrations.

At planting, fill the plug holes in the traywith dry medium and level off. Water thetray thoroughly, which causes the level ofthe medium to drop about one-third downthe plug hole.

Insert the plant carefully into the medium inthe plug using a probe instrument to createa planting hole. Do not force the plant intothe medium. From a separate bucket of wetmedium, top up the plug hole to support theplant. Compress the medium lightly aroundthe plant.

Note 1: do not plant small and large plantsin the same tray since this variability will beenhanced during subsequent growth.

Note 2: at all stages of the extraction, dip-ping and planting process, handle the plantsextremely carefully since they are fragile,brittle and easily damaged.

After planting, thoroughly water the traysand transfer them to the tray tables in theweaning house.

Figure 5.Two common weaning trays: white molded polystyrene (right) and black plastic (left).

Figure 6.Rooted banana plants in agar gel, just removed from a laboratory flask (left), separated from the gel (right) with the dipping and cleaning tank (background).

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• Step 2. Inside the weaning house

Control of environmental conditions andappropriate management of the culturaltechniques should be as follows (figure 3):

– Shade: place the trays under 80% shadenetting for the first 3 weeks (20% light trans-mission), then change the shade netting to40–50% for the next 3 weeks, or until theend of the weaning period. Arias and Val-verde recommend progressing from 70%shade for the first 3 weeks to 30% shade untilcompletion [5]. Plants could even beexposed to full sunlight to harden thembefore transferring to the nursery [1].

Note: too much light causes burning of theyoung plants, especially during the first2 weeks of weaning.

– Misting system: use a permanent auto-matic misting system to apply 10 s mistevery 20 min in hot weather and 10 s every60 min in cooler weather, via fine mist noz-zles. This should be enough to keep themedium moist in a closed greenhouse.Check the correct functioning of the mistingunit at regular intervals and ensure theyoung leaves remain wet at all times duringthe first 10 days [4]. Excessive leaf dryingcan easily be identified by curled leaves andburnt edges and tips.

Note: the rooting medium should release adrop of water when squeezed hard betweenthe fingers but should not be saturated, oth-erwise roots will suffocate and rot, and theplant will remain stunted or die. Do notwater manually with a coarse sprinkler headin an effort to keep the leaf surfaces wet inhot weather because water application isexcessive and irregular, and the mediumbecomes saturated. If the medium remainssaturated, reduce misting and/or irrigationfrequency and increase ventilation. Mistingof leaves may become less important thanirrigation towards the end of the weaningperiod [3].

Water quality is important in weaning. Makesure the conductivity of the irrigation wateris less than 30 mS·m–1 and the concentrationof total soluble salts (TSS) is less than250 mg·L–1 [6]. In addition, it is critical thesource of irrigation water should be free fromany pathogenic nematodes of banana; oth-erwise, any early nematode infestation in

weaning can be multiplied in the nursery orfield.

Note: if these salt levels are exceeded, a mar-ginal leaf burn could be experienced on thetender leaves of young plants.

– Temperature: maintain the growingtemperature between 25 °C and 32 °C at alltimes for the optimum balance betweenphotosynthesis and plant development [1].

Note: temperatures around 22 °C are opti-mal for photosynthesis and dry matterassimilation, whereas temperatures around32 °C favor plant development, stem elon-gation and leaf expansion [7]. If tempera-tures are continually below 25 °C, the plantbecomes short, squat and sturdy with shortinternodes and small leaves but with astrong root system. Physiologically theplant is healthy and strong but extra time isneeded to reach the required nursery trans-plant size. On the other hand, if tempera-tures are continually above 32 °C, thestems become elongated, with large floppyleaves and light green coloration, and theroot system may become weak. With tem-peratures approaching 40 °C, respiration isdominant over photosynthesis, reserves areused up, and the plant becomes physiolog-ically weak and prone to dying back [8]. Attemperatures above 35 °C, activate the wetwall and extraction fans, or, if there is no

Figure 7.Uniform transplants in a weaning tray, indicating effective banana plant size-grading at planting.

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wet wall, lift the sides of the weaninghouse and increase misting frequency.

– Humidity: maintain relative humidity atmore than 90% during the first week ofweaning and thereafter within the range of50% (minimum daytime humidity) to 90%(maximum night-time humidity) at all times,to reduce the rate of leaf drying [4].

Note: if humidity drops below 50%, thechances of rapid leaf drying and plant wilt-ing are increased, especially on new trans-plants. With a cover of glass or polyethylenesheeting, and frequent misting, it should bepossible to keep humidity high.

– Wind: never expose young bananaplants to wind stress at any stage during theweaning period.

Note: wind, or even a breeze, removes theboundary layer of high humidity fromaround the leaf surface. This in turn causesthe leaf surface to dry and leaf temperatureto increase, resulting in an increased risk ofleaf wilting, leaf margin scorching and plantdieback. If vents are opened in the weaninghouse walls to reduce high temperatures,take care to keep wind movement awayfrom the leaf surfaces, and increase the mist-ing frequency.

– Pest control: apply preventive sprayingfor aphids, caterpillars or leaf spot fungus,if any of these pests and diseases are prev-alent. For caterpillars, use a Bacillus thur-ingiensis formulation and, for leaf spot fun-gus, spray a systemic fungicide, at therecommended concentration. Repeat thesesprays after 6 weeks in the weaning house,or just prior to removal of plants for trans-ferring to the nursery.

Note: the last spray of fungicide protects theyoung plants from disease developmentduring early establishment in the nursery.

– Nutrition: the slow-release fertilizer orthe recommended fertigation program (seeunder weaning medium) should provide forthe nutritional needs of young plants for theduration of their stay in the weaning house.However, if leaves turn pale green or yellowin color, apply extra sprays of a NPK foliarfeed with trace elements, at 7–10-day inter-vals during weaning. For example, achelated trace element formulation whichalso contains around 10% nitrogen in the

concentrated product can be sprayed at therate of 1 g·L–1 water (i.e., 100 mg·L–1 nitro-gen in the spray solution).

Note: phytosanitary treatments indicated inthis publication are given as examples ofpossible treatments for pest control. Suchtreatments must be chosen according to thenational regulations.

Troubleshooting

Many problems can occur in the weaninghouse:

(a) The youngest leaves can be mechani-cally scarred at their tips. Causes couldbe (a) that leaves touch the flask lid; (b) thatthere was rough handling at planting.

Solution: it is superficial damage; plants willgrow out of this symptom. Do not allowplants in flasks to push against the lid. Bevery careful when removing, dipping andplanting the young plantlets.

(b) Leaves wilt and hang down; stem basesturn brown and die. This is due to overwa-tering causing root dieback.Solution: water the medium only when ithas had a chance to dry. Avoid saturation.

(c) Young leaves turn prematurely yellowand then brown. No new cigar leavesappear. The cause would be a Pythiuminfection originating from either the wean-ing medium or the irrigation water.Solution: use sterilized medium and chlo-rinated water. Alternatively, drench withsuitable fungicide, such as propamocarbhydrochloride or mancozeb/metalaxyl-m, atthe recommended concentrations.

(d) Plant growth slowing down; leavesbecome smaller and internodes shorter. Thisis due to night temperatures becoming toolow.Solution: switch heaters on if minimum tem-perature drops below 18 °C.

(e) Plants become lanky with large floppyleaves; roots are poorly developed. Thecause would be day temperatures becomingtoo high and plant development becomingtoo fast.Solution: activate the wet wall and extrac-tion fans when daytime temperature rises to35 °C; increase ventilation in the weaninghouse.

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(f) Older leaves get round necrotic spots(figure 8). This is due to leaf spot disease.Plants are left too long in the weaning phaseor there is overcrowding in trays.Solution: remove plants earlier from wean-ing. Alternatively, spray plants with systemicfungicide such as propiconazole at the rec-ommended concentration.

(g) The medium disintegrates at extraction:there is insufficient root growth, or rootsbecome stuck inside the wall of the plughole. Solution: leave plants longer to developmore roots; reduce growing temperatures tostimulate roots; spray tray plugs with 'Styro-seal' before planting. Change from polysty-rene to plastic trays.

(h) A brown ring of necrotic tissue isobserved around the leaf margin. Thiswould be a salinity burn, usually due toeither saline medium (e.g., coir), or salineirrigation water.Solution: flush salts from coir medium; useless saline medium (e.g., peat moss); use irri-gation water with conductivity less than30 mS·m–1.

(i) Plants are tall and lanky with red stems,bulbous stem bases, pale green/yellowleaves, and root-bound plugs are observed(figure 9). The cause is overmaturity due toexcessive time in weaning.Solution: do not leave plants in weaning forlonger than 8 weeks. If a delay is required,reduce tunnel temperatures.

(j) There is a plant size variability in the trayat maturity. Some plants are excessively talland some small and stunted with a poor rootsystem. Plants that are too small have to bereplanted. Causes could be (a) poor size-grading at planting; (b) overcrowding intrays; (c) uneven management throughoutthe tray.Solution: apply diligent and accurate size-grading at planting; plant fewer transplantsin the tray; ensure environmental effects areequalized across the tray.

(k) Brittle white stems of new transplantsbreak or become damaged. Causes could bethat (a) plants were too long and lanky atremoval from the flask; (b) there was roughhandling at planting into the weaningmedium.

Solution: produce shorter and thicker plantsfor transplanting; handle with care at trans-planting.

(l) White flecks and streaking appear acrossyoung leaves. This is due to variegated ormasada mutations.Solution: be vigilant in the weaning tunneland remove all such mutant plants from thesystem.

3. Typical results obtained

The standards given below refer to theexpected results from weaning of medium-

Figure 9.Banana plants left too long in weaning, resulting in overmature, senescent symptoms and root-bound plugs.

Figure 8.Mature weaned banana plant showing Cerscospora leaf spotting from overcrowded, humid conditions in the weaning house.

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sized Cavendish cultivars (‘Williams’ or‘Grand Nain’). Under normal conditions,new roots appear 4–5 days after the start ofweaning, and new leaves start to growwithin 8–10 days. When weaning is com-pleted, the following plant standards areexpected:

– Plants have an average height of 8 cmfrom the plug level to the junction of the twoyoungest leaves. Minimum height should be5 cm and maximum height 12 cm.

– Plants have at least three healthy, darkgreen leaves on the top half of the pseu-dostem and, preferably, five leaves at thetime of delivery to the nursery.

– The stem of the plant is firm and straight,not soft or flexible. The diameter of the stemat plug level is a minimum of 5 mm.

– The stem of the plant is firmly anchoredin the plug of medium and not loose or bent.

– Leaves are normal in shape and color, andthey are not folded, misshapen, senescentor flecked with any deficiency, disorder ordisease.

– When plants are extracted from the wean-ing tray, the plugs detach easily without theplant shifting its position or the roots pullingout of the medium. Roots do not grow intothe sides of the plug hole and stick there(which causes disintegration of the mediumwhen the plant is extracted).

– Upon extracting the plants, the bottomhalf of the plugs do not detach from the tophalf and the roots are totally ramifiedthroughout the medium, binding the latterinto a firm ‘root ball’.

– Upon extracting the plants, the roots arevisible on all sides and they are white andhealthy, with root hairs.

References

[1] Daniells J.W., Smith M.K., Post-flask man-agement of tissue-cultured bananas, Tech.Rep. 18, ACIAR, Canberra, Australia, 1991,9 p.

[2] Jaizme-Vega M.C., Azcon R., Responses ofsome tropical and subtropical cultures toendomycorrhizal fungi, Mycorrhiza 5 (1995)213–217.

[3] Israeli Y., Lahav E., Reuveni O., In-vitro cul-ture of bananas, in: Gowen S. (Ed.), Bananasand plantains, Chapman and Hall, London,UK, 1995, pp. 147–178.

[4] Vuylsteke D.R., Talengera D., Post-flaskmanagement of micropropagated bananasand plantains, IITA, Ibadan, Nigeria, 1998,15 p.

[5] Arias O., Valverde M., Production y variaciónsomaclonal de plantas de banano variedadGrande Naine producidas por cultivo detejidos, Asbana (San Jose, Costa Rica) 28(1987) 6–11.

[6] Lahav E., Banana nutrition, in: Gowen S.(Ed.), Bananas and plantains, Chapman andHall, London, UK, 1995, pp. 258–316.

[7] Turner D.W., The response of the plant to theenvironment, in: Gowen S. (Ed.), Bananasand plantains, Chapman and Hall, London,UK, 1995, pp. 206–229.

[8] Robinson J.C., Bananas and plantains, CABInt., Wallingford, UK, 1996, 238 p.