Project REPORTON

In vitro Seed Production and Seed Immobilization

Submitted in Partial Fulfillment of the Required Credits for the Degree of

Bachelor OF technologyIN

BIOTECHNOLOGY

SuPERVISED by Submitted by

Dr. Avinash Marwal Preeti

JV-B /10/2051

Faculty of ENGINEERING & technologyjayoti Vidyapeeth Women’s University,

Jaipur

1

CERTIFICATE

The report is hereby approved as a bonafide and creditable project work “In vitro

Seed Production and Seed Immobilization” carried out and presented by Preeti

(JV-B/10/2051) in a manner to warrant its acceptance in partial fulfillment of the

required credits for the degree of B. Tech + M. Tech in Biotechnology. However,

the undersigned do not necessarily endorse or take responsibility for any statement

or opinion expressed or conclusion drawn there in, but only approve the report for

the purpose for which it is submitted.

(…………………………) (Dr. Richa Sharma)

Supervisor (External) Supervisor (Internal)Organization Jayoti Vidyapeeth Women’s University

(Dr. Avinash Marwal)

CoordinatorDepartment of Biotechnology & Engineering,Jayoti Vidyapeeth Women’s University

(Dr. Promod Raghav)

Dean Faculty of Engineering and Technology,Jayoti Vidyapeeth Women’s University

2

ACKNOWLEDGEMENT

I express my gratitude to all those who helped me to prepare and complete my

dissertation/training/project work entitled “In vitro Seed Production and Seed

Immobilization” First of all, I convey my deep gratitude and heart full thanks to

Dr. Avinash Marwal, JVWU for his inspiration, cooperation and encouragement

for pursuing my dissertation. His valuable suggestion and guidance helped me a lot

to complete my work in this institution with in a very short period.

I render my sincere respect and heart full gratitude to Dr. Richa Sharma,

Department of Biotechnology, Jayoti Vidyapeeth Women’s University, Jaipur. I

am also thankful to all the faculty members, for their valuable suggestion towards

completing the dissertation work. I am also grateful to all my class mates, who

helped me directly or indirectly in completing my dissertation/training/project

work successfully.

Last but not least, I am really ever grateful to my parents, who remained a constant

source of encouragement and inspiration during the completion of this work

successfully in Jayoti Vidyapeeth women’s University, Jaipur.

Preeti

(JV-B/10/2051)

Department of Biotechnology

Jayoti Vidyapeeth Women’s University, Jaipur

3

DECLARATION

"I hereby declare that this submission is my own work and that, to the best of my

knowledge and belief, it contains no material previously published or written by

another person nor materials which have been accepted for the award of any other

degree or diploma of any university or institution of higher learning, except where

due acknowledgment has been made in the text.”

Place: Jayoti Vidhyapeeth, Rajasthan (Preeti)

Date: 28th Nov 2015 JV-B/10/2051

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INDEX

S.NO. TOPIC PAGE NO.

1

2

3

4

5

Introduction

Review Literature

Materials and Methods

a)Seed Viability

b)MS preparation

c)In vitro seed germination

d)Artificial seed production

Result and Discussion

References

6-8

9-12

12-13

14-17

17

18

19-22

22-26

5

INTRODUCTION

Germination testing is considered as the most important quality test in evaluating the

planting value of a seed lot. The ability of seeds to produce normal seedling and plants later on is

measured in terms of germination test. Testing of seeds under field conditions is normally

unsatisfactory as the results cannot be replaced with reliability. Laboratory methods then have

been conceived wherein the external factors are controlled to give the most uniform rapid and

complete germination. Testing conditions in the laboratory have been standardized to enable the

test results to be reproduced within limits as nearly as possible as those determined by random

sample variations.

Testing seed viability (germination) is considered as one of the secondary tasks in the

production of a cereal crop. Nonetheless, it deserves a very careful attention. Without knowing

the germination capacity of the seed to be planted, one will not be able to determine an

intelligent estimate of seeds required to ensure a adequate population of plants at the beginning

of the planting cycle. The percentage of germination of a sample taken from seeds to be planted

is an important test, but not enough sufficient.

When conventional methods produce low or no germination, in vitro techniques can

greatly enhance germination. The media composition, qualitative and quantitative aspects of

plant growth regulators play a vital role in plant in vitro studies. Therefore optimization of these

conditions is a prerequisite for in vitro plant tissue culture studies. Development of protocols for

in vitro seed germination and seedling development can be used to rapidly produce large number

of plants for conservation and restoration [1].

6

Preparation of culture media is preferred to be performed in an equipped for this purpose

compartment. This compartment should be constructed so as to maintain ease in cleaning and

reducing possibility of contamination. Supplies of both tap and distilled water and gas should be

provided. Appropriate systems for water sterilization or deionization are also important [2].

Certain devices are required for better performance such as a refrigerator, freezer, hot

plate, stirrer, pH meter, electric balances with different weighing ranges, heater, Bunsen burner

in addition to glassware and chemicals. It is well known now that mistakes which occur in tissue

culture process most frequently originate from inaccurate media preparation that is why clean

glassware, high quality water, pure chemicals and careful measurement of media components

should be facilitated [3].

A convenient method for preparation of culture media is to make concentrated stock

solutions which can be immediately diluted to preferred concentration before use. Solutions of

macronutrients are better to be prepared as stock solutions of 10 times the strength of the final

operative medium. Stock solutions can be stored in a refrigerator at 2- 4oC. Micronutrients stock

solutions are made up at 100 times of the final concentration of the working medium. The

micronutrients stock solution can also be stored in a refrigerator or a freezer until needed. Iron

stock solution should be 100 times concentrated than the final working medium and stored in a

refrigerator. Vitamins are prepared as either 100 or 1000 times concentrated stock solutions and

stored in a freezer (-20oC) until used if it is desired to keep them for long otherwise they can be

stored in a refrigerator for 2-3 months and should be discarded thereafter [4]. Stock solutions of

growth regulators are usually prepared at 100-1000 times the final desired concentration.

Plant propagation through artificial seeds broadens the horizon of plant biotechnology

and agriculture [5]. As one of the important value-added plant tissue culture products, artificial

7

seed technology can only be successful with efficient upstream production of micropropagules as

well as downstream germination protocols for high percentage of plant regeneration [6].

Production of synthetic seeds has unraveled new vistas in in vitro plant biotechnology,

such as large scale clonal propagation, delivery of clonal plantlets, germplasm conservation,

breeding of plants in which propagation through normal seeds is not possible, genetic uniformity,

easy storage and transportation etc [7] because artificial seeds technology offers several potential

advantages: (1) ease of handling, (2) low production cost, (3) ease of exchange of plant materials

between different laboratories in different counties, (4) genetic uniformity of propagated plants

(5) direct delivery to the soil, (6) shorten the breeding cycle and (7) reduction of the storage

space [8-10].

The present study was focused on the following objectives:

1. Determination of Mung bean (Vigna radiata) seed viability.

2. Preparation of various stock solutions of Murashige and Skoog (MS) medium.

3. In Vitro Seed Germination of Mung bean (Vigna radiata).

4. Artificial Seed Production through Sodium Alginate immobilization technique.

8

REVIEW LITERATURE

A study at Fairbanks, AK, was started in 1984 to determine soil seed longevity of 17

weed species. Seeds were buried in mesh bags 2 and 15 cm deep and were recovered 0.7, 1.7,

2.7, 3.7, 4.7, 6.7, 9.7, and 19.7 yr later. Viability was determined by germination and tetrazolium

tests. Seed viability data were fit to an exponential model, separately for each depth, and the

likelihood-ratio test was used to determine whether seed-viability decline was affected by burial

depth. Depth of burial had a significant effect on viability decline of prostrate knotweed, marsh

yellowcress, bluejoint reedgrass, and wild oat [11].

By 19.7 years after burial (YAB), all seeds of common hempnettle, quackgrass, wild oat,

foxtail barley, and bluejoint reedgrass were dead. Seeds of 12 other species were still viable:

corn spurry (0.1%), prostrate knotweed (0.3% at 2 cm, 0.8% at 15 cm), flixweed (0.5%),

pineapple-weed (0.6%), shepherd’s- purse (1.3%), wild buckwheat (1.5%), common chickweed

(1.6%), rough cinquefoil (1.8%), common lambsquarters (3.0%), Pennsylvania smartweed

(3.3%), marsh yellowcress (8.5% at 2 cm, 0.3% at 15 cm), and American dragonhead (62.2%).

Seed dormancy at 19.7 YAB was very low for all species (4%) except for American dragonhead,

common lambsquarters, Pennsylvania smartweed, and shepherd’s-purse, which had seed

dormancies of 100, 27, 25, and 38%, respectively. Seed longevity was not increased by cold,

subarctic temperatures [11].

The ‘algarrobo’ Prosopis flexuosa is an important food resource in the Monte Desert of

Argentina. Native, domestic and exotic mammals consume the fruit of this legume and disperse

the seed through faeces. It was analyzed the effect of different dispersal agents (cattle, horse,

European wild boar, rodents, gray fox) have on seed damage, viability and germination. Cattle

increase germination at the expense of reduced viability, whereas horses maintain viability but

9

do not contribute to a prompt germination response. Among native mammals, the gray fox

maintains seed viability without increasing germination, whereas rodents affect seed viability but

enhance germination rates. The European wild boar, however, damages all of the seeds it

consumes [12]

South American Polylepis mountain forests belong to the most endangered forest

ecosystem in the world. Reforestation measures have been strongly recommended but may be

hampered due to the very low seed germination rates reported for several Polylepis species. In

order to determine the causes behind reduced seed germination, seed viability of Polylepis

australis trees in the mountains of central Argentina were analyzed. Seeds from seven

heterogeneous areas (4-5 well-separated trees per area totaling 29 trees) with high within and

between variation in degradation status were picked. Average percentage of viable seeds was 8 -

38 and lack of an embryo was the main reason for seeds not being able to germinate. Seed

viability was positively associated with relatively undisturbed soils supporting tussock grasslands

(38.7 % of variance) and negatively associated with soil erosion (18.8% of the variance). In order

to improve seed viability, the data suggests that livestock pressure and burning practices should

be reduced, as these are the main causes for erosion and other forms of soil destruction [13].

Optimal growth and morphogenesis of tissues may vary for different plants according to

their nutritional requirements. Moreover, tissues from different parts of plants may also have

different requirements for satisfactory growth [14]. Tissue culture media were first developed

from nutrient solutions used for culturing whole plants e.g. root culture medium of White and

callus culture medium of Gautheret. White’s medium was based on Uspenski and Uspenska’s

medium for algae, Gautheret’s medium was based on Knop’s salt solution [15]. Basic media that

10

are frequently used include Murashige and Skoog (MS) medium [14], Linsmaier and Skoog (LS)

medium [16], Gamborg (B5) medium [17] and Nitsch and Nitsch (NN) medium [18].

Enhanced in vitro seed germination, seedling development protocol has been established

for conservation of very rare and globally endangered woody tree species, Butea monosperma

(Lam.) Taub. var. lutea (Witt.) Fabaceae). Mature seeds were cultured on two basic inorganic

media with full (F) or ½ strength (H) of MS supplemented with various concentrations of N6-

benzyladenine (BA, 2.22, 4.40, 6.62 and 8.40 μM) or thidiazuron (TDZ, 0.45, 2.27, 4.54, 6.80

μM) alone. The seedlings (90%) of B. monosperma var. lutea readily acclimated to greenhouse

conditions [19].

Dendrobium ovatum Lind l. is an epiphytic orchid found in the Western Ghats. A rapid in

vitro seed germination technique were used. MS, VW, B5 and KC media supplemented with

various concentrations of auxins and cytokinins were used in combination for asymbiotic seed

germination and plantlet formation. CM induced formation of PLBs which further differentiated

into plantlets. In the optimization process for phytohormones, 0.5 mg BAP/L-1 and 5 mg

NAA/L-1 favoured maximum number of plantlet formation. However, rhizogenesis was found to

be minimal in the above medium. 90 days old in vitro plantlets inside the tissue culture bottles

were seen with inflorescence production with 10-12 flowers per axis. Hardened plants were

transferred to green house after ex vitro rooting technique [20].

The somatic embryo can be encapsulated, handled and used like a natural seed was first

suggested by [21] and efforts to engineer them into synthetic seed have been ongoing ever since

[22, 23] Winkelmann et al. [24] added that the hardening of beads is generally performed in 50-

100 mM of CaCl2.2H2O, with exposure times ranging from 20 min for Cyclamen persicum to 60

min for Pelongonium horturum [25]. The highest germination rate was obtained when a 30-min

11

exposure to 50 mM CaCl2.2H2O was applied for bead polymerization, in comparison with a 10-

min exposure to 60 mM or 80 mM [26].

In Orchids, the most important cut flowers which are commercially propagated in vitro,

the best conversion to plantlets (100%) of encapsulated of Dendrobium ‘Sonia’ was observed

when 3% Na-alginate drops were hardened for 30 min in 75 mM CaCl2.2H2O [27]. Shoot tips

stored for 12 weeks and encapsulated in 3% sodium alginate prepared in distilled water without

MS medium [28]. In other studies use of meristematic shoot tips or axillary buds were used for

the production of synthetic seeds as reported for banana [29, 30].

Although it was widely accepted that orchid seeds could only be germinated with the

proper fungus, germination rates were often low and seedling death was common [31]. Bernard,

however, was successful in germinating seeds of Cattleya and Laelia in the absence of a fungus

by using salep, a powder obtained from tubers of Ophrys, a terrestrial orchid genus [32].

Oliva and Arditti [33] reported that photoperiod did not have a significant effect on

germination of several species of Aplectrum, Spiranthes, and Cypripedium. Arditti et al. [34]

reported that illumination negatively affected the germination of Calypso bulbosa and Epipactis

gigantea; however, no difference was found between illumination and complete darkness in seed

germination of Goodyera, Piperia, and Platanthera.

Rasmussen et al. [35] examined symbiotic seed germination of Dactylorhiza majalis

incubated in darkness and then placed under a 16-hour light/8-hour dark photoperiod and vice

versa. Cultures were placed under 36-watt fluorescent tubes with an irradiance of 51.2 μmol m-2

s-1. Cultures under a 16-hour light/8-hour dark photoperiod for 14 days followed by a dark

incubation for 35 days increased germination (75% germination) substantially over the control

(44% germination).

12

MATERIALS AND METHODS

Seed Viability

1. Obtain a representative sample of seed.

a. Make sure the sample chosen is representative of the seed.

b. A sample of 1500 grams should be well mixed.

c. Choose sub samples each having 10 seeds.

2. Write the details on the cover of the petri dish.

a. Use a wax pen or a thin indelible marker. Indicate the variety, the date of the test.

3. Put a filter paper in a petri dish.

a. Use the base of the petri dish.

4. Wet the filter paper.

a. Add enough clean water to wet the paper. Careful not to put much, for too much

water will make some seeds to float.

5. Put the seeds on the filter paper.

a. Distribute all the 10 seeds uniformly on the filter paper. Make sure the seeds aren`t

bunched in one location for this complicate the counting of the emerged seeds.

b. Don`t add water after placing the seeds as water drops could move the seeds.

6. Cover the petri dish.

a. Make sure you use the cover that is already marked with the information.

7. Wait the seeds to germinate.

a. Put the petri dishes in different location at different temeperature and light conditions

b. Ambient temperature is favourable for germination

c. The seeds will germinate in 4-5 days and check the seed viability.

13

Murashige and Skoog (MS) tissue culture medium (1962) Preparation

Major salts (500 mL)

NH4NO3 8.25 g

KNO3 9.5 g

CaCl2.2H2O 2.2 g

MgSO4.7H2O 1.85 g

KH2PO4 0.85 g

Bring to volume (500 mL) with distilled water and Store at 40C.

Minor salts (500 mL)

KI 0.0415 g

H3BO3 0.310 g

MnSO4.4H2O 1.115 g

ZnSO4.7H2O 0.43 g

CuSO4.5H2O 0.00125 g

CoCl2.6H2O 0.00125 g

Na2MoO4.2H2O 0.0125 g

Bring to volume (500 mL) with distilled water and Store at 40C.

Organic Supplement (500 mL)

Myo-inositol 5 g

Nicotinic acid 0.025 g

14

Pyridoxine-HCl 0.025 g

Thiamine-HCl 0.005 g

Glycine 0.1 g

Bring to volume (500 mL) with distilled water and Store at 40C.

Iron Stock (500 mL)

Sol A: FeSO4.7H2O 1.39 g (200 ml DW)

Sol B:Na2EDTA.2H2O 1.86 g (200 ml DW)

Mix solutions A and B. Bring to volume (500 mL) with distilled water and Store at 40C.

Volumes of the stock solutions (the first column from the left) required for making different

volumes of the MS medium (indicated in the first row)

200 mL 500 mL One Litre

Distilled water 150 mL 375 mL 750 mL

Major salts 20 mL 50 mL 100 mL

Minor salts 2 mL 5 mL 10 mL

Organic supplement 2 mL 5 mL 10 mL

Iron stock 2 mL 5 mL 10 mL

The method is exemplified for preparing 1.0 liter of MS basal medium.

15

1. For preparation of 1.0 liter of MS Basal medium, the above stocks solutions should be added

sequentially in about 500 ml of doubled distilled water.

2. Weight and add required quantities of sucrose (20 or 30 g) and dissolve by magnetic stirring.

3. According to the purpose of the medium growth regulator and other medium conjugates/

additives are added, and the volume of the medium is made up to 1000 ml by distilled water.

4. Adjust the pH of the medium to 5.8 using 0.1 NaOH or 0.1 N HCL before autoclaving.

5. Note that the pH meter should be calibrated by standard buffers (4.0 and 7.0) immediately

before adjusting the medium.

6. For preparing semisolid medium, add agar at the rate of 6.0-8.0 gm/l in the pH adjusting

medium, and heat until near boiling in a microwave oven or gas oven with intermittent

stirring.

7. Measured volume of semisolid media is dispenser into culture tubes, containers. Vessel using

an automatic media disperser.

8. For preparing liquid medium, pH adjusted media are directly poured in suitable containers.

9. For plating experiments, semisolid medium is poured in sterile Petri dishes.

10. Plant tissue culture media are usually autoclaved at 121 C For 20 min (15 lb in or 1.05 kg⁰

cm2).

11. Autoclaving is generally done in a horizontal or vertical autoclave.

12. Minimum time necessary for steam sterilization of media is dependent on volume of medium

per vessel and is described separately.

13. Autoclaved media are kept in ambient temperature for a day and then transferred in a dust-

free closed cabinet for subsequent use.

16

14. Semisolid medium starts drying up, and therefore should be used within a fortnight after its

preparation.

In Vitro Seed Germination

1. The mature seeds of Mung bean (Vigna radiata) were purchased from the market.

2. Collected seeds were initially washed under running water tap for 5 min and were soaked in

sterilized distilled water (SDW) at room temperature for overnight.

3. The soaked seeds were surface sterilized with 70% ethyl alcohol for 1-2 mins followed by

0.1% (w/v) HgCl2 (0.1% w/v) for 40-60 sec.

4. After each surface sterilization treatment, seeds were thoroughly rinsed 4-5 (each of 1-2 min)

with sterile distilled water (SDW).

5. After rinsing for 4-5 times with SDW, the seeds were aseptically blotted on Whatman paper

and transferred to conical flask containing 50 ml of Murashige and Skoog Media.

6. The seed explants were cultured for 1 weeks on the full strength Murashige and Skoog media

using 25 ± 2 0C with 16h photoperiod, under white fluorescent light (65 μE/m2/s).

7. After two days the germinated seeds were transferred to half strength Murashige and Skoog

Media for better root development.

8. After germination, individual seedlings after three days of culture (plantlets) were removed

from culture jars and washed thoroughly with water and transferred carefully / potted in

plastic jars / glass beaker containing sterilized soil.

17

Artificial Seed Production

1. Dissolve 9 g of sodium alginate in 300 ml of distilled water.

2. Stir until all sodium alginate is completely dissolved to avoid clump formation.

3. The final solution contains 3% alginate by weight.

4. Thoroughly suspend about 50 seeds in the alginate solution prepared in the previous step.

5. Let air bubbles escape.

6. Drip the seed-alginate mixture from a height of 20 cm into 1000 ml of crosslinking solution.

7. The crosslinking solution is prepared by adding an additional 0.05M of CaCl2 to the distilled

water.

8. The calcium crosslinking solution is agitated on a magnetic stirrer.

9. Gel formation can be achieved at room temperature as soon as the sodium alginate drops

come in direct contact with the calcium solution.

10. Wash the beads with a fresh calcium crosslinking solution and transfer to soil.

18

RESULTS AND DISCUSSION

Germination of a seed lot in a laboratory is the emergence and development of the

seedling to a stage where the aspect of its essential structures indicates whether or not it is able to

develop further into a satisfactory plant under favorable conditions in soil [36]. "These essential

structures are a well-developed and intact root system, hypocotyl, plumule and one or two

cotyledons according to the species. Seedlings cannot be evaluated in a germination test until

these essential structures are clearly identifiable and the reported percentage germination

expresses the proportion of seed which have produced normal seedlings within the period

specified for each species [37].

Figure 1: Petriplates showing Mung bean (Vigna radiata) seeds (+ & -) germination at various

temperature and light conditions.

Germination occurs under different ranges of temperatures provided the seed is given

adequate moisture. Temperature is not as critical as water requirement during the test. Seeds of

19

most of agricultural and horticultural crops germinate in the temperature range of 10 °C - 35 °C

[38]. Our results too hold the germination within the favorable range. Mung bean (Vigna radiata)

seeds showed 100% germination at Room Temperature (Both in Light and Dark condition),

Growth Chamber (15 °C) and BOD Incubator (28.6 °C) (Figure 1). Whereas Mung bean (Vigna

radiata) seeds fail to germinate at higher temperature in Incubator (50 °C) and at low

temperature in Fridge (4 °C) suggesting 0% germination rate.

Figure 2: Depicting the fast root development in liquid media.

For in vitro seed production MS media was used, supplemented with various

concentrations of nutrients. After two days seeds started germinating and no contamination was

observed. Seeds with miniature hypocotyls and plumule were transferred to half strength MS

liquid for better root development (Figure 2). Seedlings showed better root development in liquid

media as compared to semi solid media. After germination, individual seedlings after three days

20

of culture (plantlets) were removed from culture jars and washed thoroughly with water and

transferred carefully / potted in plastic jars / glass beaker containing sterilized soil (Figure 3).

Figure 3: Growth of shoot development in soil transferred seedlings

The concentration of the CaCl2 is about one fourth of the strength used for seed

immobilization. Relatively small alginate beads are preferred to minimize the mass transfer

resistance (Figure 4a). A diameter of 3-5 mm was readily achieved with a forceps. The beads

should fully harden in 1-2 hours. Individual seeds transferred carefully into potted plastic jars /

glass beaker containing sterilized soil showed germination after one week (Figure 4b).

(a)

21

(b)

Figure 4: (a) Beads showing encapsulation of seed with Alginate matrix. (b) Germinated seed via

artificial seed production technology.

This protocol may be useful in low production cost, short time conservation, ease of

handling and ease of exchange of plant materials between different laboratories in different

counties. Finally, it must be pointed that synthetic seeds composition may be differed depending

on plant type, where its endogenous construction may affected its response.

Finally, we can conclude that in vitro grown seeds and encapsulation of seeds is a

suitable system for mid-term storage of cultures since encapsulation saves space, time and

resources and it demonstrates advantages over conventional method.

22

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