Effect of Auxin (Naa/iba) and Mineral Medium on Direct Embryogenesis from Leaf Tissue

Observation of embryonic anatomy

TN 7. Callus formation from leaf fragment culture (10 x 10 mm)

TN 8. Creating embryos from callus cultured leaf fragments (10 x 10 mm) on special medium

Observation of the anatomical structure of regenerated embryos on special media

TN 9. Creating embryos from callus cultured in liquid medium

Observation of cell cluster/tissue cluster morphology in shaking liquid culture

TN 11. Creating embryos from leaf fragment callus (3 x 10 mm)

TN 10. Callus formation from leaf fragment culture (3 x 10 mm)

CONTENT 1

Creating asexual embryos

TN 12. Embryo multiplication on special medium

CONTENT 2

Asexual reproduction

Embryo multiplication in liquid medium

Indirect asexual embryogenesis through leaf fragment callus

Direct asexual embryogenesis from leaf tissue

Material

Summary diagram of research contents

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TN 23. Effect of NAA, IBA on adventitious root formation from in vitro shoots of nursery stem nodes

TN 24. Effect of NAA, IBA on adventitious root formation from shoots of in vitro stem nodes

TN 25. Effects of NAA, IBA on adventitious root formation from shoots of asexual embryonic origin

CONTENT 3

Create adventitious roots

Material

Directly induce adventitious roots from leaf tissue

Create adventitious roots from

bud

CONTENT 4

Adventitious root

2.3. Contents 1. Creating asexual embryos

2.3.1. Direct asexual embryogenesis from leaf tissue

2.3.1.1. Effect of auxin (NAA/IBA) and mineral medium on direct embryogenesis from leaf tissue

To investigate the separate effects of auxin with appropriate concentrations and mineral environment on direct embryogenesis from leaf fragments, the experiment was arranged in a two-factor manner: the factor of auxin with different concentrations; the factor of mineral environment with 4 types of environment.

Culture material: leaf pieces measuring 10 x 10 mm.

The culture medium was ½MS, MS [105], B5 [106] and SH [107] supplemented with individual auxin (NAA or IBA) at different concentrations (0, 1, 2, 3, 4, 5, 6 mg/L), containing 30 g/L of sugar (sucrose). The experiment was arranged in a completely randomized manner, with 5 samples/plate, 6 plates/replication, and each treatment was repeated 3 times. The parameters of embryogenic sample rate (%) and number of embryos/embryogenic sample at 60 NSC were recorded.

Observation of embryonic anatomy

Cut the embryo (bridge, heart, torpedo) into thin slices with a razor blade, soak the microtome in bleach until the sample is white (remove cell membrane), wash the sample with distilled water. Then, soak the microtome in 10% acetic acid solution for 10 minutes (remove bleach before staining). Perform double staining of the microtome with alum dye.

- Iodine for 15 minutes, wash the sample [43]. Observe the slide with a Leica stereo microscope (20X objective) and take pictures.

Monitoring indicators: Morphological forms of asexual embryos (globular embryos, heart embryos, torpedo embryos, dicotyledonous embryos)

2.3.1.2. Effect of auxin and BA combination on direct embryogenesis from leaf tissue

To determine the optimal BA concentration combined with auxin of appropriate concentration to induce asexual embryogenesis directly from leaf tissue.

Culture material: leaf pieces measuring 10 x 10 mm

The culture medium was a mineral medium containing auxin with concentrations that positively affected embryo formation (inheriting the results from experiment 2.3.1.1) and supplemented with BA with different concentrations (0; 0.25; 0.5; 1mg/L), 30 g/Lsucrose. The experiment was arranged in a two-factor manner: the concentration factor of auxin with concentrations that positively affected embryo formation (the above experimental results), the concentration factor of

BA (4 concentration levels). Inoculate 5 samples/plate, 6 plates/replication, each treatment is repeated 3 times. Record the indicators at 60 NSC time: embryogenic sample rate (%); number of embryos/sample.

2.3.1.3. Effects of sugar concentration and lighting conditions on direct embryogenesis from leaf tissue

Investigation of sugar concentration and suitable lighting conditions for embryo induction. The culture medium used was mineral medium with auxin concentration (NAA/IBA); BA concentration (inheriting the results from the two experiments above) supplemented with sucrose at different concentrations (10, 30, 50, 70 g/L) and cultured in light/dark conditions. In light conditions, the sample was placed under fluorescent light, illuminated for 12 hours/day, light intensity ~ 4,000 lux; in dark conditions, the sample was placed in complete darkness.

Culture material: leaf pieces measuring 10 x 10 mm.

The experiment was arranged in a two-factor model: sucrose factor with 4 different concentration levels (10, 30, 50, 70 g/L), lighting condition factor with 2 light levels (4,000 lux)/dark. Inoculate 5 samples/plate, 6 plates/replication, each treatment is repeated 3 times. Monitor and record the indicators at 60 NSC time: embryogenic sample rate (%); number of embryos/sample.

2.3.1.4. Effect of coconut water on direct embryogenesis from leaf tissue

The optimal conditions of experiment 2.3.1.3 were applied as culture medium supplemented with coconut water at different ratios of 0, 5, 10, 15, 20% (v/v); to investigate the effect of coconut water ratio on direct embryo induction from leaf tissue.

Culture material: leaf pieces measuring 10 x 10 mm.

Inoculate 5 samples/plate, 6 plates/replication, each treatment is repeated 3 times. The monitoring indicators recorded at 60 NSC include the rate of embryogenic samples (%); number of embryos/sample. The experiment was arranged completely randomly, in a single-factor manner.

2.3.1.5. Creation of complete in vitro plantlets from asexual embryos

To determine the appropriate culture environment to create complete in vitro plantlets , thereby creating plantlets with good stems, leaves, and roots to serve the purpose of planting in pots of soil.

Culture materials are seedlings with stems ~ 1 cm high, with true leaves 3 – 4 mm (originating from asexual embryos).

Separate the seedlings and culture them on petri dishes containing 30 mL of culture medium, MS or ½MS, containing 20 g/L sucrose, without adding growth promoter. Inoculate 5 seedlings/dish with 6 dishes/replication, each treatment is repeated 3 times. Observe the basic morphology,

41

Collect data on monitoring indicators after 90 days of transplanting. At 90 days, NSC measures the following monitoring indicators: Plant height (cm), number of leaves/plant, number of roots/plant; root length (mm): determined from root base to root tip; observe basic morphology.

Use the best environment of the experiment as the culture environment to create a source of healthy in vitro plantlets . Use the plantlets originating from the above asexual embryos and plant them in a triangular flask containing 50 mL of culture medium, plant 3 plants/flask, 10 flasks/replication, repeat 3 times. After 3 months, collect the plantlets to plant in pots in the nursery. The experiment was arranged completely randomly in a single factorial manner.

2.3.1.6. Planting seedlings in pots of soil

The 3-month-old seedlings were cultured in a triangular flask (from the above experimental results), removed from the triangular flask, washed with agar, preserved and ensured humidity to avoid dehydration for the plants, then planted in pots (Ø ~ 20 cm) containing clean soil of the Tribat brand (Saigon Green Biotechnology Co., Ltd., Ho Chi Minh City). The pots were placed in the nursery, planted 1 seedling/pot, 10 pots/repetition, 3 repetitions, recording the survival rate (%) and basic morphology of the seedlings at 3 weeks after planting.

Survival rate (%) = (Number of living plants/number of plants planted) x 100%

Plant trees in the nursery with ~50% shading by black net (light intensity ~8,000 lux), spray watering twice a day, temperature 30 - 32 o C, humidity 60 - 70%. Monitor, record, and evaluate the morphology of the trees 3 - 12 months after planting.

2.3.2. Indirect asexual embryogenesis through callus from leaf tissue culture

2.3.2.1. Indirect asexual embryogenesis through leaf fragment callus (10 x 10 mm)

Scar tissue formation

Culture material: leaf pieces measuring 10 x 10 mm

The samples were cultured in SH callus medium supplemented with 2,4-D at concentrations (0; 1; 2; 3 mg/L), 30 g/L sucrose. After 40 days of culture, the samples were subcultured and cultured for 20 days to form callus on the entire surface of the leaf fragment. The experiment was carried out on petri dishes, cultured 5 leaf fragments/plate, 3 plates/replication. Observe and monitor the formation of callus at 20, 40, 60 NSC.

Cut the leaf pieces bearing callus (hereinafter referred to as callus pieces) at the above 60 NSCs into pieces of size (~ 10 x 10 mm/~ 1 cm 2 ), and continue to culture for a period of time.

30 days to create embryogenic callus (KNSP). Inoculate 5 pieces of callus/disc, inoculate 6 discs/repetition, each treatment is repeated 3 times.

Monitor and record the following indicators at 30 days of age: percentage of callus samples with KNSP (%), number of callus clusters with KNSP/callus sample.

The experiment was arranged in a completely randomized single-factorial design.

Identifying callus with KNSP based on morphological characteristics, color, structure, callus with KNSP: hard, slightly yellowish white, yellow, firm structure, cluster or granular, slow growth. Callus without KNSP has spongy structure, fast growth, white or gray color, soft.

Creating embryos from callus cultured on special media

Culture material: callus clusters with KNSP

The callus clusters with KNSP obtained from the above experiment were cut into small clusters with a size of ~ 5 mm, and implanted into the embryogenesis medium, which was a culture medium supplemented with auxin and appropriate concentrations of BA (inheriting the results of the direct embryogenesis experiment). This auxin was arranged with concentrations of 1; 2; 3; 4; 5 mg/L; with 30 g/L sucrose.

Conduct 5 callus clusters with KNSP/disc, 6 discs/replication, each treatment is repeated 3 times, the experiment is completely randomized in a single-factorial design. Monitor and record data at 30 NSC with the following monitoring indicators: number of callus clusters with KNSP forming embryos, number of embryos/callus clusters with KNSP, embryo morphology characteristics.

Observation of the anatomical structure of regenerated embryos on special media

Using a razor blade, cut across the regenerated embryos from callus clusters with KNSP cultured on solid medium to create thin cell slices, then the cut samples were stained using the double staining method as above [43]. Observe the specimens using a Leica stereo microscope (20X objective) and take pictures.

Creating embryos from callus cultured on liquid medium

The experiment was conducted with the material source being callus clusters with KNSP of about 5 mm in diameter, 30 callus clusters with KNSP were implanted into a V250 mL triangular flask containing 60 mL of medium inherited from the above experimental results, each treatment was repeated 3 times. Monitor and record embryo formation at 10, 14, 30 NSCs.

Observation of cell cluster/tissue cluster morphology in shaking liquid culture

Observe and record the morphology of living cell clusters (unstained), embryogenic multicellular clusters (through staining) (at the stage of ~10 days of shaking liquid culture of embryogenic callus) under an inverted microscope with magnification of 50X, 20X, respectively.

2.3.2.2. Indirect asexual embryogenesis through leaf fragment callus (3 x 10 mm)

Callus induction from leaf explant culture (3 x 10 mm)

This experiment was designed to create a source of callus material for subsequent embryogenesis studies. The culture material used was a leaf fragment measuring 3 x 10 mm. The experiment was arranged in a completely randomized single-factorial design.

The samples were cultured on SH callus formation medium supplemented with 2,4-D at concentrations of 0; 1; 2; 3 mg/L, 30 g/L sucrose. Culture 10 samples/plate, 3 plates/replication, each treatment was repeated 3 times. Monitor and record the formation of callus, callus with KNSP at 30 NSC.

Embryo creation

Culture material: callus formed from experiment 2.3.2.2

Callus samples were cultured in an embryo-forming medium similar to the culture medium of the embryo-forming experiment in a solid medium. 5 callus samples were cultured/plate, 6 plates were cultured/replication, each treatment was repeated 3 times. The experiment was arranged in a single-factor and completely randomized manner. Monitor and observe embryo formation; collect data at 30 NSC with the following indicators: number of callus samples forming embryos, number of embryos/callus sample. Continue to observe and record embryo development at 60 NSC stage (no data collection).

2.3.3. Indicators and monitoring methods

Asexual embryos are identified as one of four morphological forms of embryo development: globular embryo, heart embryo, torpedo embryo, mature embryo (polarized form with procotyledons and proroots). Embryos are counted by observing under a magnifying glass and recording the number of embryos.

Conduct monitoring with 30 samples for each treatment.

Parameters in the direct embryogenesis experiment, monitoring and data collection at day 60 after implantation:

Embryo creation rate (%) = (Number of embryo creation/number of cultured samples) x 100% Number of embryos/sample = Total number of embryos/number of embryo creation samples

Indirect embryogenesis experiment parameters, monitoring and data collection at day 30 after implantation:

Rate of callus forming samples with KNSP (%) = ( 𝑆number of callus forming samples with KNSP 𝐾𝑁𝑆𝑃 ) x 100%

I am a single mother

Number of callus clusters with KNSP/sample = 𝑆𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚

I'm so sorry

Number of embryos/callus cluster = 𝑇𝑛𝑔 𝑠𝑝ℎ𝑖

Mother and daughter are in trouble

2.4. Content 2. Asexual embryogenesis

2.4.1. Embryo multiplication on special medium

The culture material is primary asexual embryo clusters (formed from leaf fragments).

The embryo multiplication medium was used from the results of the experiment of direct embryogenesis from leaf fragments on mineral medium, auxin type, BA concentration. This auxin type was used with different concentrations (0; 1; 2; 3; 4; 5 mg/L), 30 g/L sucrose. The experiment was arranged on petri dishes, in a single-factor and completely randomized manner. Inoculate 5 embryo clusters/dish (3 embryos/cluster), 6 dishes/replication, each treatment was repeated 3 times. Collect data on the number of embryos/cluster, embryo multiplication coefficient, observe the morphological characteristics of embryos, embryo clusters at 30 NSC. Pay attention to observe the formation of secondary embryos in parts (from stem, leaves, roots) of the primary embryo.

2.4.2. Embryo multiplication in liquid medium

2.4.2.1. Effect of growth regulators on secondary embryogenesis through shaking liquid culture

Using the results of the above experiment as a basis for the experiment to investigate the effects of DHST substances on secondary embryogenesis in liquid medium. Therefore, the culture medium was inherited from the mineral medium, the concentration levels of auxin with active embryogenesis and BA concentration; shaking liquid culture. Using a German SARTORIUS shaker, perform shaking liquid culture at 80 rpm.

The culture material was 60-day-old single embryos (from direct somatic embryogenesis from leaf tissue).

The experiment was conducted in a V250 mL triangular flask, containing 60 mL of culture medium, shaking at 80 rpm, each treatment was repeated 3 times. The experiment was arranged in a single factorial manner, implanting 30 embryos/flask. Observe, monitor the experiment, collect data on the number of secondary embryos formed, morphological characteristics and embryo color in 30 NSCs.