CHAPTER 6: SEED TECHNOLOGY

SEED TECHNOLOGY

6.1. Introduction

Characteristics of seeds such as purity, weight, dimensions, specific gravity, colour,

germination percentage and vigour have been of interest to farmers and seed scientists

alike. While the farmer’s interest stems from his need for a good crop, for the researcher it

is primarily due to its scientific relevance. Knowledge of these factors, in addition to

understanding the viability and dormancy of a particular species, also helps one in planning

suitable cultivation programmes. The present investigation looks at some of these aspects,

of the two species E. ribes and S. tetragonum, for the same reason.

The importance of seed health in cultivation has been known and highly valued since the

early times. This is evidenced by the numerous verses that are written, pertaining to this, in

the various ancient texts such as Krishi-Parashara, Kashyapiyakrishisukti, Vrikshayurveda by

Surapala, Arthashastra and Manusmriti (Nene, 2000).

Krishi Parashara, Verse 164

“Seeds which have come in contact with lamp, fire, or smoke and which are exposed

to rain or stored in a pit should always be discarded” (Sadhale, 1999)

The criteria for determining good quality seeds, namely, viability, purity, vigour, seed health

and noxious seed contamination, that are considered important today, were of significance

in the ancient times too. However, standard testing procedures to test good seeds were

probably not available then (Nene, 2000). Measurement standards were also mostly not

uniform. Units such as anguli and hasta were used, which could vary from person to person.

With regard to seed viability, farmers in ancient India have traditionally used the easy and

simple ‘floating test’ to differentiate between viable and empty seeds. Winnowing was

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another method used to separate out the empty seeds. Both these methods, although still

in use, are crude methods of determining seed viability.

Although these are easy methods that have been successfully used in India since ancient

times, they are not universally accepted. In order to maintain uniformity in measurement

standards of seed parameters throughout the world, the International Seed Testing

Authority (ISTA) has provided standardized definitions and methods to be used for

evaluating seeds with respect to the above mentioned characteristics (ISTA, 2004). This not

only helps in comparison between samples from different regions, but also in easy

international trade of seeds. In the current study, seed and fruit characteristics of E. ribes

and S. tetragonum have been calculated as per ISTA standards. Seed viability of the two

species was also determined during the study.

6.1.1. Seed and fruit morphology

Details of the fruit and seed morphology are provided in Chapter 1 of this thesis. In the

present study, details of seed weight, dimensions and moisture content of seeds of E. ribes

and S. tetragonum collected from different provenances were determined.

6.1.2. Seed viability

‘Germinability’ of a seed is its ability to produce ‘normal’ seedling, whereas ‘viability’ of a

seed denotes whether it is ‘alive’ or ‘dead’ (Gosling, 2003). Although germination test is the

most reliable test for determining what percentage of a seed-lot will produce seedlings, a

viability test is also acceptable, as it indicates what percentage of the seeds is more likely to

produce seedlings. The viability test becomes inevitable, especially when the time taken for

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germination is very long, due to any of the dormancy mechanisms, or when the seed

involved is ‘recalcitrant’ (Gosling, 2003).

Cut test, x-ray test, topographical tetrazolium (TTZ) test, excised embryo test, vital stain test,

etc. are some of the techniques used to measure viability of seeds. These tests, by different

indications, give an estimate of the percentage of live seeds in a seed lot. However, these

tests have several limitations, some of them either under-estimating or over-estimating the

germination rate (Chacko et al., 2002). Whereas the cut test and the X-ray test assess the

viability of a seed by observation of internal structures of the seed by cutting open the seed

and x-ray radiography respectively, the TTZ test and vital stain test make use of chemicals to

identify living cells within the seed to ascertain its viability. The excised embryo test, on the

other hand, involves extraction of whole, healthy, fully grown embryos from the seed and

their incubation on moist filter paper.

Three methods for determining seed viability, viz., TTZ test, Cut test and Clearing technique

were carried out during the present study for calculating the viability percentage of the two

species.

TTZ test: The TTZ test is considered to be the ideal method for assessing seed viability when

seeds are dormant, slow germinating, recalcitrant, or when a quick estimate of germination

potential needs to be made (ISTA, 2004). The basic principle of this test is that the enzyme

dehydrogenase, present only in living cells, is able to reduce the soluble tetrazolium salt into

insoluble, red coloured formazan. During the present investigation, both the species E. ribes

and S. tetragonum were subjected to TTZ test to determine the percentage of seed viability.

Cut test: The cut test is the quickest test to determine the viability of seeds, although it is a

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crude method of analysis of viability. It can also be used to determine the stage of maturity

(for collection) and the efficiency of the processing method used. It does not require many

specialized equipments (Gosling, 2003). However, it is a subjective test which determines

the percentage of viability by ocular observation of the condition of the essential tissues

inside the seed (Chacko et al., 2002; Willan, 1985). S. tetragonum was subjected to this test

during the present study.

Clearing technique: The clearing technique has been used by taxonomists and

morphologists to observe whole mount of plant parts, since many decades (Gardner, 1975).

One of the methods of clearing involves making the internal tissues translucent, without

changing its structure, by the use of certain chemicals (Herr, 1993). Chemicals like Herr’s

fluid, lactic acid and sodium hydroxide (NaOH) are used for clearing internal tissues (Herr,

1971; Buechler, 2004; Lux et al., 2005). In the present study NaOH was used as the clearing

agent to ascertain the presence of embryo within the seeds of E. ribes.

6.2. Materials and methods

6.2.1. Seed and fruit morphology

Fruits of E. ribes and S. tetragonum were collected and their weight, dimensions and

moisture content were measured. Fruit/seed weights were measured using a digital

weighing balance [Essae Electronic Weighing Balance Model PG1000]. All lengths were

measured using a measuring tape, with minimum accuracy of 1mm. Other dimensions, like

breadth and diameter, were measured using vernier calipers by the standard procedures.

Moisture content was calculated using oven dry method (ISTA, 2004).

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6.2.2. Viability testing

TTZ test:

Preparation of stain solution: 1g of 2,3,5 triphenyl tetrazolium chloride was weighed out

using a digital weighing balance and dissolved in 1l distilled water to prepare a 1% colourless

solution of the salt (ISTA, 2004). Use of buffer solution for preparation of the solution is

recommended, if the pH of the solution is not between 6.5 and 7.5. During the present

study, as distilled water was used for preparation of the solution and its pH was within this

range, buffer solution was not made use of for preparation of the stain solution.

Preparation of the seeds for the TTZ test: 100 seeds in 4 replicates of the selected species,

E. ribes and S. tetragonum, were soaked in distilled water for 18 hours. These hydrated

seeds were then cut into two and placed in petridish (ISTA, 2004). Soaking in water as well

as cutting the seeds ensures maximum penetration of the stain.

Following this, the distilled water was drained out and stain solution was poured over the

soaked seeds, such that they were immersed completely in stain solution. They were then

placed in the dark, at ambient temperature. The seeds were observed, for staining, every

hour for a period of 8 hours.

Cut test:

100 seeds in 4 replicates of S. tetragonum collected from Devala were taken and using a

blade the seeds were cut, along the central ridge. The contents of the seeds were inspected

for observation of cotyledons, embryo and other essential organs (Ooi, 2007; Chacko, 2009).

Number of seeds with embryo was recorded and images taken using [Olympus DP72]

camera, with Imagepro Version 6.0 software.

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Clearing technique:

100 seeds of E. ribes in 4 replicates were soaked in 10% NaOH taken in a petridish (Buechler,

2004). The petridish was sealed using paraffin film and incubated in a hot air oven at 60°C

for 1 hour. After cooling, the seeds were observed under the stereomicroscope [Olympus

DX41] (Gardner, 1975). Number of seeds with embryo was recorded and images taken using

camera [Olympus DP72], with Imagepro Version 6.0 software.

6.3. Observations

6.3.1. Seed and fruit morphology

During the present investigation, it was observed that fruits of E. ribes have a moisture

content of 60-70% at the time of dispersal, which reduces to 15-20% upon drying. Thus, they

lose more than 75% of their moisture content when dried (Figure 6.1). The diameter of the

fruits also reduces from 0.35-0.60cm to 0.30-0.55cm upon drying, i.e. size of the fruit

reduces by 10-15% when dried. Seed weight also reduces considerably from 12,000-20,000

fruits/kg when fresh to 40,000-50,000 fruits/kg upon drying (Table 6.1).

The fruits and seeds of S. tetragonum are dry at the time of dispersal (Figure 6.2). Therefore,

dimensions and moisture content of the fresh as well as dry fruits are more or less similar.

With respect to seeds also the same was observed (Table 6.1). The seeds, being papery and

light, are dispersed by wind.

6.3.2. Viability testing

TTZ test: The seeds of E. ribes and S. tetragonum were unstained at the end of 8 hours. It

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Figure 6.1: Fruits of E. ribes (a) Fresh; (b) Dry

Figure 6.2: (a) Fruits of S. tetragonum; (b) Seeds of S. tetragonum

Table 6.1: Fruit and seed details of E. ribes and S. tetragonum

Particulars E. ribes S. tetragonum

Fruit Seed Fruit Seed

Weight Fresh 12000-20000/kg 100000-110000/kg 250-300 pods/kg 65000-77000/kg

Dry 40000-50000/kg 129000-139000/kg 250-300 pods/kg 65000-77000/kg

Dimension

(cm)

Fresh 0.35-0.60* 0.15-0.30

* 20.0-50.0 X 0.5-1.0

# 2.02-3.13 X 0.40-0.54

#

Dry 0.30-0.55* 0.14-0.24

* 20.0-50.0 X 0.5-1.0

# 2.02-3.13 X 0.40-0.54

#

Moisture

(%)

Fresh 60-70 20-25 50-60 10-13

Dry 15-20 12-17 40-50 10-13

No. of seeds

-- 1 per fruit 20-50 per fruit

[ *diameter ;

#length X breadth ]

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was observed that, with respect to E. ribes, although the cotyledons took up very light stain,

the embryo remained unstained even after 170 hours (Figure 6.3). With respect to seeds of

S. tetragonum also it was seen that neither the cotyledons nor the embryo took up any stain

(Figure 6.4).

Figure 6.3 (a), (b), (c): E. ribes seeds subjected to TTZ test

Figure 6.4 (a), (b), (c): S. tetragonum seeds subjected to TTZ test

Cut test: Out of the 100 seeds in each replicate of S. tetragonum seeds, replicates 1 and 2

had 12 and 10 seeds, respectively, with healthy and mature cotyledons and embryos (Table

6.2). In one of the replicates (replicate 4), 20 out of the 100 seeds were good, healthy seeds;

while in replicate 3 only 2 seeds were healthy. On an average 11% seeds were observed to

be viable. Most of the remaining seeds were totally empty, with neither cotyledon nor the

embryo being present, while a few seeds were seen to contain shriveled, blackened

structures which are probably rudimentary cotyledons.

Although, occular observation was sufficient to observe the presence or absence of

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cotyledons and embryos in the seeds of S. tetraognum, for better understanding of the

internal structures, the seeds were observed under the stereomicroscope (Figure 6.5).

Figure 6.5: Cut test of S. tetragonum seeds (a) with healthy cotyledons; (b) without

cotyledons; (c) with rudimentary cotyledon/embryo

Figure 6.6: Clearing technique of E. ribes seeds (a) whole embryo; (b) part of the embryo showing hypocotyl; (c) part of the embryo

showing epicotyl and cotyledons

Table 6.2: Result of Cut test on S. tetragonum seeds

Replication no.

No. of seeds with cotyledon/embryo

No. of empty/ half empty seeds

Total no. of seeds

1 12 88 100

2 10 90 100

3 2 98 100

4 20 80 100

Avg 11 89 100

Clearing technique: When seeds of E. ribes were subjected to the clearing technique, it was

observed that about 83% of seeds possessed visibly mature and healthy embryos (Table

6.3). The cotyledons, plumule and radicle are clearly distinguishable in the embryo (Figure

6.6). Some of the embryos were also found floating in the clearing solution, after becoming

detached from the seed.

Table 6.3: Result of Clearing technique on E. ribes seeds

Replication no.

No. of seeds with embryo

No. of seeds without embryo

Total no. of seeds

1 83 17 100

2 83 17 100

3 85 15 100

4 80 20 100

Avg 82.75 17.25 100

6.4. Discussion

6.4.1. Seed and fruit morphology

E. ribes is a woody climber that produces globose, dark purple to black drupes in terminal or

axillary panicles. The fruits resemble black pepper when dry and have been reported as

being used as its adulterant (Nayak et al., 2009). They have high moisture content at the

time of dispersal and are shed at the slightest disturbance from the panicles. Each fruit

contains a single hard-coated seed, which has characteristic white patches on the surface.

S. tetragonum is a large-sized tree that produces linear, elongated, twisted, 4-angled,

drooping capsules in loose panicles towards the tip of branches. Each pod is spirally twisted

and consists of 20-50 seeds arranged in the depressions of a central septum. It is a dry

dehiscent fruit that opens along the margins of the fruit walls when mature, releasing the

winged seeds. The fruit walls and the central septum are left behind on the tree. These do

not fall off easily and are visible for one year or more on the tree.

6.4.2. Viability testing

TTZ test: The tetrazolium test is a quick and useful tool for assessing the viability and vigour

of seed lots (Patil and Dadlani, 1993; Vankus, 1997; Pant et al., 1999; Schmidt, 2000; Chacko

et al., 2002). It is based on the reduction action of the respiratory enzyme dehydrogenase.

This enzyme is present only in living cells and is capable of converting the soluble 2,3,5

triphenyle tetrazolium chloride salt into insoluble 2,3,5 triphenyle formazan. Thus, the

colourless tetrazolium salt that enters the living cells is converted into red coloured

precipitate, imparting a deep red colour to living cells. Dead cells that do not contain the

enzyme are unable to form formazan and therefore, remain colourless. The intensity and

pattern of staining, which is species specific, is assessed to determine the viability of the

seed. A seed is considered viable if its essential structures like embryo, meristems and most

part of the cotyledons are stained. On the contrary, if these structures remain unstained,

the seed is considered as non-viable. In addition to the staining, tissue soundness is also to

be considered to determine the viability of a seed. Standardized protocols of TTZ test for

different species have been worked out by various authors (Gupta et al., 2010; Zeng et al.,

2014; Cui et al., 2014).

In the present study, it was observed that none of the embryos or cotyledons of E. ribes

took up considerable stain. No staining was observed even when the embryos were placed

in the TTZ solution for a period of 170 hours, which leads one to the presumption that none

of the seeds are viable. However, it has been observed during the germination trials that

when seeds were subjected to specific pretreatments, they germinate (details in Chapter 7).

This disparity in the results between the TTZ and germination tests could be either because

the methodology for TTZ test, followed during the present study, is not suitable to

determine the viability percentage of E. ribes or this species does not respond favourably to

the TTZ test.

Similarly, the embryo as well as the cotyledon of S. tetragonum remained unstained even

after soaking the seeds in the stain for 170 hours. However, as observed for E. ribes,

germination trials in this species too have shown positive results (details in Chapter 7). Thus,

the procedure for TTZ test adopted during the present study is not suitable for this species

too.

Viability percentage of E. ribes and S. tetragonum, using TTZ test, has not yet been reported.

Therefore, it can be concluded that, with respect to both the selected species, the standard

procedure of testing viability using TTZ is not suitable. Either the procedure needs to be

suitably modified for each of the species, or other suitable tests are to be identified to

determine the seed viability of these species. The need for considerable expertise in

conducting the test as well interpretation of the results of the TTZ test has been reported in

earlier studies too (Chacko et al., 2002; Gosling, 2003). The practical difficulty encountered

while attempting to separate the seed coat from the wings with respect to S. tetragonum

seeds is also a deterrent for conducting this test.

Cut test: The cut test is an effective means of assessing viability percentage of a seed lot in

many plant species (Ooi, 2007; Chacko, 2009). It neither involves sophisticated equipments

nor is there a necessity for skilled manpower for interpretation of the result. It is based on

visible signs of a healthy seed. Seeds that are empty, underdeveloped or damaged by insects

or any other means are classified as non-viable, and the rest are classified as viable seeds.

Thus, it is more farmer-friendly and can be adopted by lay persons too.

In the present study, the viability percentage of S. tetragonum could not be assessed using

the TTZ test. From the cut test it was observed that 11% of the seeds possessed visibly

healthy cotyledon and embryo. This result is comparable with the result of the germination

trials conducted on seeds subjected to specific pretreatments. (details in Chapter 7). The

results of cut tests carried out on seed lots collected from Itanagar and the FRLHT campus

also were in comparison with the results of the germination tests of seeds collected from

these provenances (Annexure 2). Thus, the cut test is a reliable assessment of the viability of

the seed lot. Moreover, the difficulty in separating the wings from the seed coat, required

for carrying out the TTZ test in this species, is not a hindrance as it is not a prerequisite for

carrying out the cut test.

The seeds of E. ribes being very small and with a hard seed coat, it was practically not

possible to subject it to the cut test. Therefore, the clearing technique was carried out for

observation of whole seed mount of this species.

Clearing technique: The clearing technique has been used for observation of sections as

well as whole plant parts such as stem, root, leaves, etc. for decades (Lux et al., 2005).

Observation of whole embryo using this technique has also been reported by de Vega and

de Oliveira (2007). In the present study, clearing technique, using NaOH as the clearing

agent, was applied to observe whole seed mount of E. ribes to ascertain the presence of

healthy embryo within the seeds. It was observed that about 83% of the seeds possessed a

single, linear, curved and centrally located embryo, with clearly distinguishable plumule,

radicle and cotyledonary leaves (Figure 6.6). The occurrence of an endospermic seed with a

linear embryo in the family Primulaceae has been reported earlier by Hartmann et al.

(2004). Also, the viability percentage observed by this method is comparable with the

results of the preliminary germination trials conducted during this study, which has shown

that 83% seeds of this species germinate after suitable pretreatment (details in Chapter 7).

Therefore, this test can be considered as an effective test to determine the viability

percentage of seeds for this species.

6.5. Conclusion

6.5.1. Seed and fruit morphology

As the seeds of E. ribes are high in moisture content at the time of dispersal, they have to be

properly dried and stored in airtight containers, so that seeds retain maximum viability.

With respect to S. tetragonum, although the seeds have low moisture content, it would be

ideal to dry the seeds and store them in airtight containers to reduce loss of viability.

6.5.2. Viability testing

The percentage of viability of a seed lot is of interest to the farmers and cultivators, as it

reveals the number of live seeds in it. It gives an indication of the germination capacity of a

seed lot, and thereby the required rate of planting to produce a good stand. The TTZ test

conducted during the present study was not successful in determining the viability

percentage of either E. ribes or S. tetragonum seeds collected during the study (Table 6.4).

The reason for this could be either because the seeds were not responsive to TTZ salt or

because the protocol was not suitable. The cut test, on the other hand, was found to be

effective in calculating the viability percentage of S. tetragonum seeds. This is an easy

technique, by which the viability percentage can be accurately calculated. It neither involves

technical expertise nor complicated interpretation skills; hence, the cut test can be carried

out by lay persons too. However, the cut test could not be used to determine the

percentage of viability in E. ribes, owing to the small size and hard seed coat of its seeds.

Table 6.4: Summary of response of E. ribes and S. tetragonum to different viability tests

Sl. no. Viability test E. ribes S. tetragonum

1 TTZ test

2 Cut test

3 Clearing technique

Therefore, the clearing technique was used for this species and was found to be effective. A

functional laboratory, with basic equipments such as an oven and a microscope, are

required for this test. However, this test is also simple and easy, and the embryo is visible

following clearing. Hence, it can be used to determine the viability of this species. Thus, this

study has lead to identification of the cut test for S. tetragonum and the clearing technique

for E. ribes as reliable methods for determination of seed viability. However, as both the cut

test and the clearing technique are destructive tests, further studies are to be conducted to

identify non-destructive methods to identify viable seeds from a seed lot.