Micropropagation Systems of Feijoa (Acca sellowiana (O. Berg) Burret)

45

Maurizio Lambardi et al. (eds.), Protocols for Micropropagation of Selected Economically-Important Horticultural Plants, Methods in Molecular Biology, vol. 994, DOI 10.1007/978-1-62703-074-8_4, © Springer Science+Business Media New York 2013

Chapter 4

Micropropagation Systems of Feijoa ( Acca sellowiana (O. Berg) Burret)

Miguel Pedro Guerra , Gabriela Claudia Cangahuala-Inocente , Lirio Luiz Dal Vesco , Rosete Pescador , and Clarissa Alves Caprestano

Abstract

Acca sellowiana (O. Berg) Burret sin. Feijoa sellowiana (Myrtaceae) is a semiwoody fruit species native to South Brazil, Uruguay, and Argentina; edible fruits are tasty. The naturally occurring populations in Santa Catarina State show high variability in fruit size, color, and other features. A breeding program launched in 1990 resulted in the release of four Brazilian commercial varieties. The conventional clonal propagation methods of this species, such as cutting and grafting, have shown low ef fi ciency. Therefore, tissue culture techniques were developed for mass propagation. This chapter describes several protocols based on organo-genesis and somatic embryogenesis. Additional techniques including synthetic seed technology and tem-porary immersion system are also described.

Key words: Arti fi cial seed , Feijoa sellowiana , Mass propagation , Myrtaceae , Organogenesis , Somatic embryogenesis , Tissue culture , Zygotic embryo

Acca sellowiana (O. Berg) Burret sin. Feijoa sellowiana (Myrtaceae) is native to the southern region of Brazilian Plateau and also found in Uruguay and Argentina ( 1 ) . It naturally occurs in the highlands of the Atlantic Forest Biome and shows high genetic variability ( 2, 3 ) and produces tasty fruits ( 4 ) called “Goiaba-Serrana” in Brazil, and Feijoa and Pineapple Guava in other parts of the world. Although this plant has been cultivated commercially in New Zealand, Australia, USA, and some European countries since the beginning of the last century, it has recently been domesticated and commercially cultivated in Brazil ( 5, 6 ) . The conventional methods of clonal propagation, such as cutting and grafting, are inef fi cient ( 7 ) . Considering this, tissue culture techniques were

1. Introduction

46 M.P. Guerra et al.

developed for mass propagation of A. sellowiana by organogenesis ( 8 ) . Among salt formulations woody plant medium (WPM) was the most responsive ( 9 ) . It was also shown that the basal culture medium devoid of plant growth regulators (PGR) was effective in the induction of organogenesis ( 10 ) . The production of adventi-tious shoots from young leaves was also reported ( 11 ) . Culturing of nodal segments and apical meristems showed low rates of bud neoformation ( 12 ) . Nodal segments of the genotype 101 showed high regeneration rates on WPM medium devoid of PGR. Ex vitro rooting was obtained with 100 mM indole-3-butyric acid (IBA) treatment for 60 min ( 13 ) .

Since the induction of somatic embryogenesis in these species, protocols have been improved ( 14 ) . The number of somatic embryos has increased on liquid plant medium (LPm, see ref. ( 15 ) ) containing 4 mM glutamine (Gln) ( 16 ) . The half strength Murashige and Skoog (MS, see ref. ( 17 ) ) culture medium supple-mented with 0.5 μ M benzylaminopurine (BAP) ideally germinated somatic embryos into plantlets. However, somatic embryogenesis was genotypic dependent ( 18 ) , and 2,4-Dichlophenoxiacetic acid (2,4-D) pulse treatment induced somatic embryogenesis. It has been postulated that genetic reprogrammation of explant tissues culminating with the induction of somatic embryogenesis is associ-ated with the methylation of nuclear DNA modulated by the 2,4-D supplemented to the culture medium ( 19 ) . Cangahuala-Inocente et al . ( 20 ) showed high rates of embryogenetic induction (100%) and number of somatic embryos/explant in response to glutamic acid (Glu) and 2,4-D. The quality of somatic embryos was improved with abscisic acid (ABA) ( 21 ) . Filament of stamens inoculated in LPm supplemented with several types and levels of PGR resulted in the development of somatic embryos ( 22 ) .

A schematic representation of the modulation of somatic embryo-genesis in A. sellowiana is presented in Fig. 1 . The differences among the protocols are mainly attributed to the genotype-dependent response of A. sellowiana to tissue culture techniques.

1. Potted plants as a source of explants. 2. Benlate ® . 3. Ethanol 70% (v/v). 4. Sodium hypochlorite (NaOCl) with 5% of active Cl. 5. Tween 20. 6. Sterile distilled water. 7. Ascorbic acid. 8. Citric acid.

2. Material

2.1. Micropropagation by Organogenesis

2.1.1. Surface Sterilization of Source Material

474 Micropropagation of Feijoa

9. Sterile Whatman ® No. 1 fi lter paper. 10. Mercury (II) chloride (HgCl 2 ).

1. Media based on Murashige and Skoog—MS salts ( 17 ) . See media formulations in Table 1 .

2. Media based on WPM salts ( 9 ) (see Table 1 ). 3. Morel’s vitamins ( 23 ) (see Table 1 ). 4. Staba vitamins ( 24 ) (see Table 1 ). 5. Sucrose. 6. Activated charcoal. 7. PGR BAP, IBA, 1-Naphtaleneacetic acid (NAA), Gibberellic

acid (GA 3 ), 6-( γ , γ -Dimethylallylamino) purine (2-iP), N6-furfuryladenine (Kinetin), and 4-Amino-3,5,6-trichloro-2-pyridinecarboxylic acid (Picloram).

8. Difco Agar.

1. Tissue culture facilities—instruments (scalpel, forceps, spirit burner to fl ame sterilize instruments), laminar fl ow cabinet, cul-ture room, pH meter, Phytotron autoclave, test tubes, bottles.

2. Sodium hydroxide (NaOH) (0.5 N). 3. Hydrochloric acid (HCl) (0.5 N).

2.1.2. Culture Media Composition

2.1.3. General Supplements

Fig. 1. Modulation of somatic embryogenesis in Acca sellowiana.


Table 1 Saline and vitamin formulations of basal culture media

MS ( 17 ) WPM ( 9 ) LPm ( 15 )

mg/L mg/L mg/L

Macronutrients Ammonium nitrate NH 4 NO 3 1650 400 1200 Potassium nitrate KNO 3 1900 – 1900 Calcium chloride CaCl 2 × 2H 2 O 440 96 180 Calcium nitrate Ca(NO 3 ) 2 – 556 – Potassium phosphate KH 2 PO 4 170 170 340 Potassium sulfate K 2 SO 4 – 990 – Magnesium sulfate MgSO 4 × 7H 2 O 370 370 370

FeEDTA solution Sodium EDTA Na 2 EDTA × 2H 2 O 37.2 37.3 19.0 Ferrous sulfate FeSO 4 × 7H 2 O 27.8 27.8 14.0

Micronutrients Zinc sulfate ZnSO 4 × 7H 2 O 8.6 8.6 2.88 Manganese sulfate MnSO 4 × H 2 O 16.9 16.9 1.69 Potassium iodide KI 0.83 – 0.75 Boric acid H 3 BO 3 6.2 6.2 0.63 Sodium molybdate Na 2 MoO 4 × 2H 2 O 0.25 0.25 0.025 Cupric sulfate CuSO 4 × 5H 2 O 0.025 0.25 0.0025 Cobalt chloride CoCl 2 × 6H 2 O 0.025 – 0.0025

Vitamins Morel ( 23 ) mg/L Staba ( 24 ) mg/L

Myo-inositol 100 2000 Thiamine HCl 1.0 1.0 Nicotinic acid 1.0 2.0 Pyridoxine HCl 1.0 2.0 Pantothenate 1.0 1.0 D-Biotin 0.01 1.0 Choline chloride – 1.0 4-aminobenzoic acid – 0.5 Folic acid – 0.5

Add sucrose (30 g/L). Adjust the pH of the medium to 5.8 with 0.5 N NaOH, autoclave at 121 °C for 16 min

1. Trays containing 72 cells (60 cm 3 each). 2. Substrate: mixed commercial substrate of vermiculite and car-

bonized rice coat (1:1; v/v). 3. Plastic box covered with glass.

1. Immature seeds. 2. Mature seeds. 3. Floral buds. 4. Ethanol 70% (v/v).

2.1.4. Ex Vitro Rooting

2.2. Micropropagation by Somatic Embryogenesis

2.2.1. Explant Sterilization


5. Sodium hypochlorite (NaClO) with 5% of active Cl. 6. Sterile distilled water.

1. Media based on LPm salts ( 15 ) . See media formulations in Table 1 .

2. Morel’s vitamins. 3. Sucrose and maltose 4. Phytagel ® . 5. Difco Agar. 6. Glutamine (gln). 7. Myo-inositol. 8. Glutamic acid (glu). 9. Plant growth regulators (BAP, GA 3, 2,4-D, 2-ip, Picloram,

kinetin). 10. Fluridone .

1. Tissue culture facilities—instruments (scalpel, forceps, spirit burner to fl ame sterilize instruments), laminar fl ow cabinet, culture room, pH meter, Phytotron autoclave, test tubes, Petri dishes, nipple- fl asks.

2. Stereomicroscope. 3. Celetor apparatus (Sigma). 4. Steward apparatus. 5. Carmine. 6. Acetic acid 45% (v/v). 7. Evan’s Blue. 8. Re fl ux condenser. 9. Slides and coverslip. 10. Light microscope. 11. RITA ® bioreactor. 12. Substrate PlantMax ® . 13. Vermiculite. 14. Trays containing 72 cells (60 cm³ each). 15. Plastic box covered with glass.

1. Torpedo-staged somatic embryos. 2. Media based on LPm salts. 3. Morel vitamins. 4. Sucrose. 5. Difco Agar. 6. Activated charcoal.

2.2.2. Culture Media Composition

2.2.3. General Supplements

2.3. Synthetic Seeds


7. Plant growth regulators (BAP, GA 3 ). 8. Petri dishes (9 × 120 cm diameter). 9. Alginic acid Sodium Salt from brown algae (Fluka). 10. Calcium chloride (CaCl 2 ). 11. Potassium nitrate (KNO 3 ). 12. Distilled water. 13. Tissue culture facilities—instruments (scalpel, forceps, micropi-

pette, tips).

An important step in the establishment of in vitro regeneration protocols is the choice and maintenance of the mother plant in adequate nutritional and sanitary conditions. For A. sellowiana , selected mother plants are cultivated in pots with 50 L capacity, containing PlantMax ® as substrate. The plants are maintained in a Phytotron at 25 ± 2 °C under 16 h photoperiod, and 200 μ mol/m 2 /s photosynthetic photon fl ux supplied by cool-white fl uorescent lamps (Sylvania ® ) and high pressure sodium vapor lamps (Empalux ® —VST) (see Fig. 2a , b). The plants are watered every day and irrigated monthly with a commercial nutritional solution.

1. WPM or MS ( 17 ) basal media are supplement with Morel’s vitamins or Staba vitamins and 3% sucrose.

2. Add other components, such as growth regulators, as required. Add the correct volume of growth regulators from 1,000 μ M stock solutions to obtain the required fi nal concentrations (see Note 1 ).

3. Adjust pH to 5.8 using 0.5 N HCl or 0.5 N NaOH. 4. Add 0.65% Difco Agar. 5. Sterilize the media by autoclaving at 121 °C for 15 min. 6. Store the autoclaved media at room temperature in the dark

for a maximum of 1 month. 7. The cultures are maintained in culture room at 25 ± 2 °C, pho-

tosynthetic photon fl ux of 40 μ mol/m 2 /s, 16 h photoperiod, and relative humidity of 60 ± 5%.

1. Two months before initiation of in vitro cultures, selected mother plants (see item 3. 1 ) are drastically pruned to promote the new growth. Additionally, the pruned plants are sprayed with 0.1% Benlate ® .

3. Methods

3.1. General Procedures for Mother Plants

3.2. Organogenesis

3.2.1. Culture Conditions

3.2.2. Shoot Regeneration


2. One month before the in vitro introduction, selected mother plants are transferred to Phytotron ® at 25 ± 2 °C under 16 h photoperiod, and 100 μ mol/m 2 /s photosynthetic photon fl ux supplied by cool-white fl uorescent lamps (Sylvania ® ) and high pressure sodium vapor lamps (Empalux ® —VST). Reduce light intensity to stimulate etiolation of new sprouts. The matrix plants were sprayed three times every 5 days with 1 g/L Benlate ® .

3. Collect sprouts with two pairs of leaves and the apical mer-istem, place in sealed plastic bags, and transport immediately to the laboratory.

Fig. 2. Nodal cultures of A. sellowiana : ( a ) mother plants kept in chamber room with 16 hour photoperiod photosynthetic photon fl ux of 100 μ mol/m 2 /s and 60 ± 5% relative humidity. Note the new elongated sprouts with light green leaves; ( b ) mother plants kept in chamber room with 16 hour photoperiod photosynthetic photon fl ux of 200 μ mol/m 2 /s and 60 ± 5% relative humidity. Note the full expanded dark green leaves; ( c , d ) microcut-ting in WPM medium after 15 days; and ( e ) after 30 days.


4. Wash them in tap water for 10 min. 5. In laminar fl ow cabinet minicuttings with nodal segments are

treated with 70% ethanol for 1 min. In sequence they are sub-mitted to a double disinfestation with 5 and 2.5% NaOCl solu-tion and Tween 20, each for 5 min. Rinse explants three times in sterile distilled water containing 250 mg/L citric acid and 250 mg/L ascorbic acid for preventing oxidation.

6. The leaves of the explants are carefully removed with a scalpel. 7. Culture explants in test tubes containing 15 mL basal gelled

WPM medium (see Fig. 2c , d). 8. Explants multiply every 30 days by the same medium of induc-

tion (see Fig. 2e ).

1. In laminar fl ow cabinet, sterile apical buds of 1–1.5 cm long with 70% ethanol for 20 s and after with 1% NaOCl for 10 min. Rinse three times in sterile distilled water.

2. Culture explants in test tubes (22 × 150 mm) containing 15 mL basal gelled MS medium supplement with 0.2% activated char-coal, 2.0 μ M BAP, 0.3 μ M GA 3 , and 0.05 μ M NAA.

3. Subculture every 30 days in the same induction medium.

1. Dip minicuttings (3–4 cm) from rejuvenated shoots of selected mother plants in an antioxidant solution (citric/ascorbic acid 360:284 μ M).

2. In laminar fl ow cabinet, sterile minicuttings are dipped in etha-nol (70%) for 1 min, and then submitted to a double disinfes-tation with 0.15% HgCl 2, 1% NaOCl solution, and Tween 20, each for 5 min. Rinse explants three times in sterile distilled water. Reduce microcuttings to 1–1.5 cm long and dip them again in the antioxidant solution.

3. Transfer them to bottles containing 30 mL gelled MS medium supplemented with 13 μ M BAP and 0.3 μ M GA 3 .

4. Subculture every 30 days to the same culture medium described in Subheading 3.2.4 , step 3.

1. In laminar fl ow cabinet, sterile the seeds with 0.5–1.0% NaOCl for 20 min. Rinse explants three times in sterile distilled water.

2. Germinate sterilized seeds in bottles (300 mL) containing 30 mL basal gelled WPM medium supplemented with 0.15% activated charcoal.

3. The germination is undertaken in culture room at 25 ± 2 °C, with 16 h photoperiod, photosynthetic photon fl ux of 40 μ mol/m 2 /s, and 60 ± 5% relative humidity.

4. Shoots from 6- to 8-week-old seedlings are used to initiate cultures for shoot multiplication.

3.2.3. Shoot Regeneration from Apical Meristem

3.2.4. Shoot Regeneration from Nodal Segment

3.2.5. Shoot Regeneration from Seedlings


5. Nodal segments (1–1.5 cm long) are inoculated in test tubes containing 15 mL of basal gelled medium WPM supplemented with 5 μ M Kin.

6. Alternatively: (a) The culture medium may be PGR free. (b) Two nodal segments (~2.5 cm of long) are inoculated in

WPM basal liquid medium with 20 μ M IBA for 6 days. After induction, the explants are transferred to basal gelled medium WPM without auxin (see Note 2 ).

(c) Nodal segments (1–1.5 cm long) are inoculated in test tubes containing 10 mL of basal medium WPM supple-mented with Staba vitamins, 3% sucrose, and gelled with 0.8% Difco Agar (see Note 3 ).

7. Transfer microcutting containing two nodal segment (~2.5 cm long) to a PGR-free basal solid medium or alternatively, to ex vitro substrate in Phytotron ® (see Note 4 ).

1. Subculture shoots with three nodal segments to test tubes con-taining 15 mL gelled ¼ MS basal medium supplemented with 1.2 μ M IBA and 0.15% activated charcoal, and grow up to 30 days.

2. After 30 days, transfer plantlets to test tubes containing 15 mL basal gelled MS medium with 0.15% activated charcoal.

1. Plantlets are transferred to trays with 72 cells fi lled with PlantMax ® after 30 days of rooting. The trays are then placed inside a plastic box covered with glass to keep a saturated humidity microenvironment.

2. The trays are placed in Phytotron ® adjusted to 27 ± 2 °C, pho-tosynthetic photon fl ux of 200 μ mol/m/ 2 /s, 16 h photope-riod, and relative humidity of 80 ± 5%.

1. The basal LPm is supplemented with Morel’s vitamins. Add 0.2% (w/v) Phytagel ® .

2. Follow all other steps according to Subheading 3.2.1 . 3. Maintain cultures in the dark culture room at 25 ± 2 °C during

the induction phase.

1. Wash immature fruits with water and commercial neutral soap and remove fruit epicarp.

2. In laminar fl ow chamber, the seeds are disinfested with 70% ethanol for 1 min; 1% NaOCl for 20 min, and then rinse three times in sterile distilled water.

3. With the aid of a stereomicroscope, excise immature zygotic embryos and inoculate them in test tubes containing 20 mL

3.2.6. Rooting

3.2.7. Plantlet Acclimatization

3.3. Somatic Embryogenesis

3.3.1. Culture Conditions

3.3.2. Somatic Embryogenesis from Immature Zygotic Embryos


basal solidi fi ed LPm supplemented with 10 μ M 2,4-D and 4 mM Gln. The cultures are maintained in the culture room at 23 °C in the dark.

4. After 120 days in culture, isolate and transfer somatic embryos (SE) ranging from the torpedo to the cotyledon stages, to half-strength MS medium, containing 0.5 μ M BAP. During the mat-uration and conversion stages of SE, keep the cultures in culture room with a photosynthetic photon fl ux 40 μ mol/m 2 /s pro-vided by cool white lamps and a 16 h photoperiod at 23 °C.

1. Place mature seeds in 0.5% NaClO solution for overnight. 2. In laminar fl ow cabinet, the seeds are disinfested with 1%

NaOCl for 20 min, and then rinse three times in sterile dis-tilled water (see Fig. 3a ).

3. Remove two external integuments of seed with scalpel under the stereomicroscope (see Fig. 3b ).

4. Culture zygotic embryos in test tubes containing 15 mL basal gelled LPm supplemented with sucrose 3% 20 μ M 2,4-D, 0.1 g/L myo-inositol, and 8 mM Glu.

5. Uncoil of zygotic embryos is observed after 5 days (see Fig. 3c ; Note 5).

6. The identi fi cation of embryogenic cultures is performed by the double staining with acetic carmine and Evan’s Blue technique, according to Gupta and Durzan ( 25 ) (see item 3 . 3 . 5 ; Fig. 3d ).

7. After 30 days, globular stage somatic embryos are observed on the surface of the cotyledon leaf (see Fig. 3e ).

8. The subsequent developmental stages, heart, torpedo, and cot-yledon, are observed after 45 days in culture. An induction rate of 100% can be obtained after 90 days in culture (see Fig. 3f ).

1. Floral buds of the A. sellowiana are collected before anthesis and sterilized as described in item 3. 2 . 3 .

2. After dissecting the bud tissues in aseptic chamber, the stamens are excised and inoculated in test tubes containing 15 mL of basal gelled medium LPm supplemented with sucrose 3% 4 mM Gln, 10 μ M Picloram, and 1 μ M Kin. Cultures are kept in culture room, in the dark at 27 °C for 60 days.

3. Embryogenic calluses are transferred to Petri dishes (15 × 100 mm) containing 25 mL of basal gelled culture medium LPm sucrose 3% supplemented with 5 μ M Picloram and 1 μ M Kin. Cultures are kept in culture room in the dark at 27 °C for 60 days.

4. Embryogenic calluses are subcultured to Petri dishes containing 25 mL of basal gelled LPm culture medium supplemented

3.3.3. Somatic Embryogenesis from Mature Zygotic Embryos

3.3.4. Somatic Embryogenesis from Stamens


with 1 μ M Picloram and 0.5 μ M Kin. The cultures are kept in culture room at 27 °C during 60 days.

5. For the scaling-up of suspension cultures, transfer 1 g fresh mass of embryogenic culture in 1,000 mL nipple- fl asks, con-taining 100 mL basal liquid LPm supplemented with sucrose 3% 1 μ M 2,4-D and 1 μ M 2-iP.

6. The nipple- fl asks are placed on a rotating orbital shaker (Steward Apparatus) at 1 rpm and incubated at 27 °C in the dark.

7. Cell fractions higher than 74 μ m are collected using stainless steel tissue sieves (Celetor apparatus, Sigma) and re-inoculated

Fig. 3. Induction and development of A. sellowiana somatic embryogenesis: ( a ) seeds extracted from mature fruits; ( b ) excised embryos after the integuments are removed; ( c ) uncoil of zygotic embryos after 5 days in culture; ( d ) embryogenic cells stained with acetocarmine after 15 days in culture ( arrow : somatic pro-embryo); ( e ) globular and heart-staged somatic embryos on the surface of cotyledonal leaf after 30 days; and ( f ) Torpedo and cotyledonary-staged somatic embryos after 90 days in culture. Bars: a , b : 0.1 cm; d : 100 μ m.


on Petri dishes, containing 25 mL of gelled LPm PGR-free culture medium.

8. For the maturation step, the cultures are subcultured to Petri dishes containing 25 mL of gelled LPm PGR-free culture medium sucrose 3%.

1. The quality of cultures is evaluated by double staining under light microscope based on acetocarmine and Evan’s Blue staining ( 25 ) .

2. An aliquot of cells is placed on a watch glass. 3. A drop of 1% acetocarmine (w/v) (see Note 6 ) is added to the

sample for 1 min. 4. Excess is removed with the aid of toilet paper. 5. Drop 0.05% Evans Blue (w/v) (see Note 7 ) for 1 min. 6. Remove again the excess with the aid of toilet paper. 7. Drop 1 mL of sterile distilled water. 8. Drop with a pipette an aliquot on a slide glass, then visualize in

the light microscope (see Note 8 ; Fig. 3d ).

1. Select torpedo-staged somatic embryos (see item 3 . 3 . 3 ) for conversion to seedlings (see Fig. 4a ).

2. Inoculate them in Petri dishes containing 25 mL of basal gelled medium LPm supplemented with 3% sucrose 0.15% activated charcoal, 0.5 μ M BAP, and 1 μ M GA 3 (see Fig. 4b ) for 30 days.

3. The converted plantlets are inoculated in test tube containing 15 mL of basal gelled LPm culture medium supplemented with 3% sucrose and 0.15% activated charcoal (see Fig. 4c ).

4. The plantlets are maintained in culture room with the photo-synthetic photon fl ux of 40 μ mol/m 2 /s provided by cool white lamps, relative humidity of 60 ± 5%, and 16 h photoperiod.

3.3.5. Cytochemical Procedures

3.3.6. Conversion of Somatic Embryos to Plantlets

Fig. 4. Conversion of Acca sellowiana somatic embryos. ( a ) Somatic embryos torpedo-staged (Bar, 0.25 cm); ( b ) the somatic embryos, inoculated in Petri dishes containing basal gelled medium LPm supplemented with 0.15% activated charcoal, 0.5 μ M BAP, and 1 μ M GA 3 ; ( c ) seedlings after 60 days.


Synthetic, arti fi cial, or somatic seeds are analogous seeds to the true or botanical seed. Each somatic embryo is encapsulated in a calcium alginate bead, which protects against mechanical damages during the storage and sowing ( 26 ) . In order to improve the con-version of the synthetic seeds, the supplementation of nutrients has been evaluated, including PGRs and addition of other substances to the alginate matrix ( 18, 20 ) .

Basically, a mixture of somatic embryos and sodium alginate is used for the encapsulation. Pick up a single somatic embryo together with sodium alginate with an Eppendorf pipette tip and release in CaCl 2 solution. For germination, immerse synthetic seeds in KNO 3 (100–200 mM) solution for 2 min to soften the capsule (see Fig. 5 ).

1. Select torpedo-staged somatic embryos (see item 3 . 3 . 3 ; Fig. 4a ). 2. Preconvert them in Petri dishes containing 25 mL of basal

gelled medium LPm supplemented with 0.15% activated char-coal, 0.5 μ M BAP, 1 μ M GA 3 , and sucrose 3% (Preconversion medium), for 15 days before encapsulation ( see Fig. 4b ).

3. The synthetic seeds are established by capturing preconverted somatic embryos in a capsule with 1% sodium alginate diluted in the medium ½ LPm salts supplemented with 0.15% acti-vated charcoal, 0.5 μ M BAP, 1 μ M GA 3 ( see Fig. 6a ) , and then

3.4. Synthetic or Arti fi cial Seeds

3.4.1. Synthetic Seeds from Somatic Embryogenesis

ab d

ce

g

h Ca+ Ca+

Ca+

i

f

Fig. 5. Schematic illustration for A. sellowiana synthetic seed production. ( a ) Petri dish containing torpedo and cotyledon-ary-staged somatic embryos; ( b ) 1% sodium alginate (w/v) diluted in LPm culture medium (gelled matrix); ( c ) pipette; ( d ) somatic embryos dipped in 1% sodium alginate (w/v) diluted in LPm culture medium (gelled matrix); ( f ) capture of somatic embryos embedded in the gelled matrix; ( g ) which are dipped in a 50 mM CaCl 2 solution; ( h ) detail of capsule complex-ation; and ( i ) conversion of synthetic seeds.


releasing the capsule in a solution of 50 mM CaCl 2 for 20 min ( see Fig. 6b ).

4. The capsules containing preconverted embryos are placed in bottles (200 mL) containing 30 mL of water gelled with 0.7% Difco Agar ( see Fig. 6c ).

5. The synthetic seeds are maintained in culture room at 25 ± 2 °C with the photosynthetic photon fl ux of 40 μ mol/m 2 / s pro-vided by cool white lamps, relative humidity of 60 ± 5%, and 16 h photoperiod.

6. Alternatively the capsules are sowed into trays with 72 cells (16 cm 3 each) containing a mixture of vermiculite and Plantmax ® 1:1 (v:v) and then maintained in acclimatization room ( see Fig. 6d , e ).

7. The trays are kept in Phytotron ® adjusted to 25 ± 2 °C, 16 h photoperiod with a photosynthetic photon fl ux of 200 μ mol/ m 2 /s. The trays are placed inside a plastic box cov-ered with glass to allow light entry and to reduce water exchange. Wet periodically the plantlets germinating capsules with a 20% solution of LPm salts (see Note 7 ).

A temporary immersion system (RITA ® ; recipient for automated temporary system) is a bioreactor, developed by Teisson and Alvard ( 27 ) . This system is relatively simple and easy to use, allowing con-tact between all parts of the explants with the nutrient solution,

3.5. Liquid Culture in Temporary Immersion System

Fig. 6. Acca sellowiana synthetic seeds. ( a ) Preconverted somatic embryos complexed with 1% sodium alginate in solution of 50 mM CaCl 2 for 20 min; ( b ) synthetic seeds with 1.5% activated charcoal; ( c ) conversion of synthetic seeds after 30 days; ( d ) ex vitro conversion of synthetic seeds placed into trays containing as substrate a mixture of vermiculite and Plantmax ® 1:1 (v:v), and ( e ) acclimatization of plantlets derived from synthetic seeds.


together with the renewal of the atmosphere by ventilation of culture promoted by the air compressor (see Fig. 7 ). Several authors reported many advantages including improved micropropagule qual-ity, reduced consumables costs, reduced labor costs ( 28, 29 ) , bet-ter leaf development, and reduced hyperhydricity ( 30 ) . Further, plants from the temporary immersion system have been found to be more suitable for acclimatization and development towards photoautotrophy ( 29 ) .

1. Embryogenic cultures (see item 3 . 3 . 3 ), containing embryos in different developmental stages are inoculated in RITA ® appa-ratus containing 200 mL of basal liquid medium LPm supple-mented with sucrose 3% 0.5 μ M BAP, 1 μ M GA 3 , and 0.05 μ M Fluridone.

2. The cultures are kept in this condition during 30 days for the conversion of embryos into plantlets.

3. After the plantlets are inoculated in test tube containing 15 mL of LPm basal medium, supplement with 3% sucrose, 0.15% activated charcoal, and gelled with 0.7% Difco Agar.

4. The plantlets are maintained in culture room at 25 ± 2 °C with 40 μ mol/m 2 /s photosynthetic photon fl ux of provided by cool white lamps, relative humidity of 60 ± 5%, and 16 h photoperiod.

3.5.1. Conversion of Somatic Embryos in System RITA ®

Fig. 7. A temporary immersion system RITA ® developed by Teisson and Alvard ( 27 ).


1. Remember 0.001 M = 1 mM = 1,000 μ M. To determine how many g/L are needed for 0.001 M concentration, fi rst, look up the molecular weight of the PGR. Consider as example the Kinetin. Its molecular weight is 215.2, then a 0.001 M solu-tion (1,000 μ M) consists of 0.2152 g/L of solution. 1,000 μ M=0.001 M

1

l

Mmol

m

V=

×

M = molarity (in g/L); m 1 = m ass of solute (in grams); mol = molar mass of solute; V l = liter of solution.

110.001g/L 0.2152g

1L 215.2

mm= ∴ =

×

2. After 30 days the mean microcutting rooting is 68.9%. 3. After 15 days, the microcuttings are subcultured to the same

medium composition. 4. Select microcuttings with two nodal segments (~2.5 cm long).

The basal portion of microcuttings is treated with 100 μ M IBA for 60 min and transplanted into trays containing substrate composed of a mixture of vermiculite and carbonized rice coat 1:1 (v:v). During acclimatization phase, the plantlets should be sprayed every week with a solution of ¼ WPM salts. Thirty days in this condition the plantlets are 45.3 mm long with a mean root number of 11.3.

5. The zygotic embryos are excised in aseptic chamber and inocu-lated in test tubes containing 15 mL of basal gelled medium LPm supplemented with 20 μ M 2,4-D and 4 mM Gln. After 2 weeks the cultures are transferred to the basal medium LPm PGR-free. The induction of somatic embryogenesis occurs directly from the cotyledonary surface of the zygotic embryos starting from 30 to 45 days. Two weeks of 2,4-D shock results in 93.7% of embryogenic induction after 12 weeks.

6. Acetocarmine 2%: Dissolve 2 g carmine in 100 mL acetic acid 45% (v/v). Boil in re fl ux condenser by 3 h. Cool in room tem-perature and fi lter.

7. To Evan’s Blue: Dissolve 1 g Evan’s Blue in 100 mL distilled water.

8. Comment : small isodiametric cells with high nucleoplasmic, few small vacuoles stained in red are embryogenic cells.

4. Notes


References

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Micropropagation Systems of Feijoa (Acca sellowiana (O. Berg) Burret)

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