6

2. Review of Literature

Seeds are the principal means of regeneration of most woody and climbing plants.

They serve as the delivery system for the transfer of genetic materials from one generation

to the next. The part of a plant life cycle that involves flowering, seed formation,

maturation, dissemination, and germination is a complex—yet fascinating—chain of

events, many of which are still poorly understood. However, some knowledge of these

events is necessary for successful collection and utilization of seeds for reforestation

(Franklin Bonner, 1996).

The patterns of growth and the timing and amount of flowering have important

repercussions for the recruitment of plant species through their effects on reproductive

process such as pollination and timing of seed dispersal (Wiegand et al., 1995).

Phenology is the study of bud growth, leaf flush, flowering, anthesis, fruiting and

leaf fall in relation to seasons or years with reference to climatic factors. Each species has

its own calendar of events to perform these cyclic developmental changes (Nagarajan, 2004).

Sagreiya (1942) highlighted the techniques for collecting phenological record of shrubs

and ornamental trees.

Blatter (1906) correlated the flowering season with the climate and Harper (1906),

while describing the phytogeography of Georgia, USA provided a phenogram to

consolidate all the phenological features. A phenological variation is observed with in a species

at levels like geographical races, ecotypes and among individuals (Mandal et al., 1994).

Generally, aspects such as leaf flushing, flowering and fruiting in tropical trees are

fairly well documented (Sun et al., 1996). Investigations suggest that a strong seasonality

7

exists in tree phenology within sub-tropical forest ecosystems and are set to initiate during

the transition of dry to wet seasons so that rainfall facilities recruitment of plants through

germination (Kikim and Yadava, 2001).

The synchronization of flowering with leaf flushing seems to be related to

moisture, temperature and photoperiod (Sun et al., 1996) with cool and dry winter

responsible for maximum leaf drops whereas increased temperature, during warm and dry

periods induces leaf flushing and flowering in most species (Kikim and Yadav, 2001).

In case of semi-evergreen plants they are known to increase their plasticity through

phenological asynchronicity among individuals and species (Williams et al., 1997;

Devineau, 1999). In dry environments, hererogencity and periodicity of water availability

have been demonstrated as crucial factors in phenological rhythms of tree communities

and populations (Seghieri et al., 1995). At individual and community level, the quantum

of flowering is inconsistent, abundant in some years and meager or absent in others. This

variation in flowering and fruiting patterns affect the degree of genetic variability in each

species (Radhamani et al., 1998).

The intensity of flowering in trees is known to vary among years (Appanah, 1990).

In the neotropics, gregarious flowering is generally an inter-specific phenomenon; with

much less synchronization among species, and flowering is more evenly distributed on a

regular basis (Frankie et al., 1974). In Madhuca indica, very low rate of seed set under

normal environmental condition due to development of one ovule out of eight per fruit

were observed (Kuruvilla, 1989).

Borua and Bezbaruah (1993) studied the morpho-phenology of ten horti-

silvicultural trees of tea plantations of North Eastern India. Mahadevan (1991) pointed out

8

the phenological characters of some forest tree species and correct period of seed

collection in Tamil Nadu. Arjunan et al., (1995) worked out the phenology of some woody

angiosperms of Coimbatore District. They also observed the period of leaf fall, leaf bud

break, flowering and fruiting in twenty five woody species.

Fruits collected from the ground are contaminated with soil borne pathogens

(Gray, 1990). When seeds are collected from the ground, there is a risk of including

overripe fruits. So preferably the ground should be cleared under the trees, covered by

clean tarpaulins during collection to avoid inclusion of old seeds (Thomsen, 2000).

Morphological characters of many seed have been studied in various autecological

investigations, including Anogeissus latifolia, Diospyros melanoxylon and Terminalia tomentosa

(Chaurey, 1953); Shorea robusta (Jain, 1962) and Anogeissus latifolia (Joshi, 1962).

According to Mayer and Poljakoff-Mayber (1963) the size and shape of seeds is

variable depending on the structure and form of the ovary, the environmental conditions,

under which plant is growing during the period of seed germination. Athaya (1985)

reported the seed characteristics of 16 tree species common to tropical dry deciduous

mixed forests of Central Indian region. The bigger seed size and more weight in Bauhinia

variegata, Diospyros melanoxylon, Pongamia pinnata and Terminalia species resulted in

large amount of response of food material to the growing embryo in comparison to the

smaller seed size and lesser weight in Anogeissus pendula and Mitragyna parviflora,

which showed better help in dispersal.

The fruit of neem is an ellipsoidal drupe, about 1.25 cm long. Its colour is green

and turns yellow on ripening. The fruit is generally one seeded and rarely they two seeded

9

(Anon., 1978). Wiersum and Rahman (1982) reported the number of seeds per kg in

Acacia auriculiformis between 53,000 to 62,000 seeds.

Pathak et al., (1974) found an increasing trend in respect of heavy seeds towards

the southern latitude and an occurrence of very heavy seeds in Australia source Leucaena

leucophloea only. The seed length, breadth and the thickness also followed the same

pattern as that of the seed weight and in the field the heavier seeds registered higher

emergence.

Bagghi and Emmanuvel (1984) reported the pod length and seed count in Albizia

lebbeck. Mathur et al., (1984) reported the number of seeds per kg and the length, breadth

and thickness of seeds of Acacia nilotica. Martin and Rashid (1982) reported the

morphological characters of the fruit and seeds of seven Albizia species. They found that

the fruit size and weight were highest in Albizia lebbeck and highest number of seeds per

fruit was in Albizia falcataria.

Ahmed and Grainge (1986) after a deep study on economic importance of

Azadirachta indica seed found that it contains many promising substances that are

effective against many important pests such as ringworm and skin diseases such as

Scrofula and Indolent.

Seed longevity (i.e. the period of survival) primarily is dependent on storage

temperature, seed moisture and oxygen availability. Stress created by climatic conditions

before physiological maturity also plays a role (Harrington, 1973). Lower the values of

these factors, the longer the conservation period of the seeds. Moisture content has long

been used to relate seed longevity.

10

Based on seed moisture content, seeds have been classified as orthodox seeds and

recalcitrant seeds (Roberts, 1973). Orthodox seeds can be dried to low moisture content

without damage to the seeds and can be maintained satisfactorily ex-situ over long term in

appropriate environments. On the other hand, recalcitrant seed storage is problematic.

They suffer desiccation mortality, if allowed to dry below critical moisture levels. Short-

term storage is the best that can be achieved with these seeds.

Seed longevity varies greatly among species. It may also vary among accessions

within a species because of differences in genotype and provenance. Influences of

provenance on potential longevity result from the cumulative effect of environment during

seed maturation, harvesting, drying and the pre-storage environment, and the time of seed

harvest, duration of drying and the subsequent period before seed is placed in store (Hong

and Ellis, 1996).

Physiological causes for reduced storability may be ascribed to failure of

accomplishing essential stages of late maturation events like incomplete embryo

development, inadequate protection from desiccation or inadequate formation of storage

proteins or chemical compounds necessary for storability (Hong and Ellis, 1990; Vertucci

et al., 1996). The immature seeds tend to be more prone to processing damage

(mechanical or heat) and may be more susceptible to infection (Schmidt, 2000).

Desiccation tolerance can decline before shedding in both intermediate (Ellis et al.,

1991) and recalcitrant (Fu et al., 1994) seeds. This may result from the initiation of

germination processes, which can reduce storage potential (Hong and Ellis, 1992).

The seed deterioration was found in some forest species to be fast in stored seeds

of low initial viability, hence good quality seeds should be stored for long period (Holmes

11

and Buszewick, 1985; Magini, 1962). For long-term genetic conservation, it is

recommended that no seeds should be accepted for storage which has an initial viability

less than 85% of that considered typical for the species (IBPGR, 1976). The higher the

initial viability of the seed lots that enters into storage, the longer the seed viability under a

given storage environment (Schmidt, 2000).

Plant growth hormones or bio-regulators or growth regulators are organic

compounds, which are known to regulate the physiological processes of plants even in

small concentrations. The growth regulators viz., auxins, gibberellins, cytokinins, abscisic

acid and ethylene have been found to induce and accelerate early biochemical events

during germination (Khan, 1977), which are very crucial and vital to increase the rate and

percentage of germination. Some seeds which have little resistance to germination

responded well to soaking for 24 hours in water at ambient temperature (Kemp, 1975).

Gray and Steckel (1977) found out that GA3 and Indole butyric acid were very

effective in alleviating suboptimal temperature effects on seed germination in Zizyphus

mauritiana. The growth regulators like GA3 and IBA were reported to stimulate and

promote seed germination and seedling vigour in a wide variety of tree crops (Khan, 1977;

Webb and Wodd, 1980; Gopikumar and Moktan, 1994; Bhattacharya et al., 1991).

Seed germination and seedling growth of Cassia obtusifolia in response to IAA,

GA3, Kinetin (KN), Chlorocholine chloride (CCC), Maleic hydrazide (MH) in different

concentration showed that IAA and KN in low concentrations enhanced both germination

and growth. GA3 promoted germination, while CCC increased germination at low

concentrations only (Singh and Murthy, 1987a). In the case Cassia fistula, GA3 was found

to be promotory for germination and for an increase in all the growth parameters. Other

12

hormones also were either found to be totally promoted or promotory at lower

concentrations (Singh and Murthy, 1987b).

Virendra Singh (1989) studied the effect of GA3 on seed germination of Picea

smithiana. The study revealed that lower gibberllin concentrations were required at longer

soaking periods. The exclusive role of gibberllins as the primary germination promoting

hormone had been demonstrated in Platycodon grandiflorum and Lavandula angustifolia

(Karssen et al., 1989).

Singh (1989a) stated that growth regulators such as coumarin, MH and

Chlorocholione chloride (CCC) increased the growth parameters viz., fresh and dry

weights of root and lateral root formation in Cassia sophera seedlings. Inhibitors of the

development of lateral roots were observed under GA3 treatment. Singh (1989b) reported

IAA to be the best hormone for development of lateral roots. Thus IAA, GA3 and Kinetin

in different concentrations, especially in the lower concentration can be applied to seeds

before sowing for realizing increase in all the growth parameters and also to obtain

maximum seed germination percentage in Cassia glauca. Ramamoorthy et al., (1989)

stated that hot water treatment of Derris indica seeds for 15 min. improved the speed of

germination and viability. The growth regulators gibberellic acid, thiourea and kinetin

showed either marginal or no response. Increase in membrane permeability and activation

of hydrolytic enzymes were cited to be the possible reasons for the improvement by hot

water treatment. Unnikrishnan and Rajeeve (1990) reported a significant increase in

germination of teak seeds treated with IAA (300 ppm) over control and GA3.

Misiha and El-Ashry (1991) studied the effect of GA3 and H2SO4 on seed

germination of Magnolia grandiflora. The highest germination percentage was achieved

13

by soaking in 1000 ppm of GA3. GA3 treatment especially at 1000 ppm increased seedling

length, fresh weight and seed carbohydrate content. Youseef et al., (1991) studied the

effect of growth regulators on the germination of different Acacia species. High amounts

of endogenous growth promoters (GA3 and IAA) and low amounts of endogenous growth

promoters (ABA and phenols) promoted germination percentage and rate. Eucalyptus

hybrid seeds gave better germination and vigour index when treated with GA3 at 100 ppm

(Bhattacharya et al., 1991). Virendra Singh (1992) found that the highest germination

percentage was obtained by treating the spruce seeds with 1.5 x 10¯ 5 M concentration of

kinetin solution after 72 hours of soaking.

Rajendra Kumar Sharma et al., (2006) reported that Rheum australe seeds were

treated with 1000ppm GA3 for 24 hours increased the germination up to 89 percentage.

Strawberry seeds treated with GA3 300 ppm showed highest germination (34%) compared

to control (22%) (Demirson et al., 2010).

Mcneil and Duran (1992) reported that when Plantago ovata seeds were treated

with GA3 and GA4 at higher concentrations, both had increased germination percentage

and GA4 was found to be more effective than GA3. Growth regulators, GA3, kinetin and

CCC significantly increased neem seed germination percentage, root and shoot lengths at

both 200 and 400 ppm (Ponnusamy, 1993). Kumaran et al., (1994) reported that in

Azadirachta indica, seed germination was enhanced by 200 ppm kinetin. Seedling vigour

and number of leaves were greatly enhanced by seeds soaked in 400 ppm Chlorocholine

chloride (CCC).

Gopikumar and Moktan (1994) noticed the highest germination in Cassia fistula

and Bauhinia purpurea with IBA at 300 ppm. Gibberellic acid and indole butyric acid at

14

300 ppm concentration yielded the best germination and seedling survival in Atropha

belladonna (Bisht Kediyal, 1995). According to Virendra Singh et al., (1995), seeds of

Quercus leucotrihophora treated with GA3 solution 500 ppm for 24 hours gave maximum

germination. GA3 400 ppm and 200 ppm recorded maximum collar diameter and above

and below ground biomass. A maximum germination (89.5%) was obtained by treatment

of Atropa belladonna seeds with GA31000ppm (Elena Genova et al., 1997). Seeds of

Ferula assafoetida treated with GA3 2000 ppm at 4º C obtained 91.6% germination

(Zare et al., 2011).

Chauhan and Nautiyal (2007) noticed GA3 at 100 ppm treatment seeds favoured

highest germination (90%) to Nardostachys jatamansi species. According to Ali Rong Li

et al., (2007), seeds of Pedicularis species treated with GA3 solution 500 ppm for 24 hours

gave maximum germination (96%). The seeds of Hippophae salicifolia treated with

200ppm GA3 improve germination up to 83% (Airi et al., 2009). Mohammad Sedghi et

al., (2010), reported that the soaking of Andrographis species seeds in 200 ppm GA3

improved germination (89%). A maximum germination (85%) was obtained by treatment

of Polygonatum rumicifolium seeds with GA3100ppm (Vinay Prakash et al., 2011).

Masilamani and Dharmalingam (1995) reported that three months old seeds of

silver oak treated with 250 ppm GA3 solution for 24 hours gave 43 percent germination

against 9 percent in control. Improvement in vigour in this treatment was six fold higher

than the control. Gopikumar and Kunhanu (1995) found that 250 ppm IAA and 250 ppm

GA3 were equally effective in enhancing the length and number of roots of some selected

tree species. Sivagnanam (1995) reported that the soaking of Azadirachta indica seeds in

100 ppm IBA improved emergence percentage and seedling vigour under nursery

15

conditions. According to Nidhi Srivastava et al., (2011), seeds of Aconitum heterophyllum

treated with IAA 500ppm recorded high seed germination (97.17%).

Simancik (1996) studied the influence of seed treatment with gibberellin on the

physiology of germination in ash tree seeds (Fraxinus excelsior). He observed that

reduced and total sugar contents in the embryo and endosperm increased with an

increasing suitability of seed pre-treatment. Palani et al., (1996) noticed in Albizia lebbeck

that 100 ppm IAA registered higher germination than water soaking. Ilango (1997)

reported that germination and vigour of Tamarindus indica was significantly improved by

IAA at 100 ppm. The 100 ppm of IBA soaking proved superior to other presowing

treatments for enhancing root and shoot lengths, number of leaves and total dry weight

both in Albizia lebbeck and Tamarindus indica.

Maitreya Kundu et al., (1997) studied the effect of pretreatments on seed

germination of Alstonia scholaris. Results indicated that soaking in IAA (200 ppm) out

performed others recording 3 and 5 fold increase in germination and vigour over control

followed by GA3 (100 ppm) wet treatment, water soaking for 24 hours and continuous hot

water (50°C) soaking for 30 minutes.

Ilango et al., (1998) reported that seeds of Albizia lebbeck soaked in 100 ppm IBA

for 12 hours produced the longest shoot and root and higher total seedling dry weight.

In Emblica officinalis (Amla), maximum germination (82%) was attained with kinetin

100 ppm. The treatment has also augmented the Emblica officinalis seedling length and

vigour index (Murugesh et al., 1998). Vanangamudi et al., (1998) reported that seeds of

Syzygium cuminii (Jamun) soaked in 100 ppm IBA for 12 hours improved the germination

and vigour. In Tamarind (Tamarindus indica), IAA and succinic acid proved superior to

16

others in enhancing seed germination and seedling vigour. Venkatesh et al., (1998)

reported that seeds of Pongamia pinnata soaked in 100 ppm IBA for 12 hours recorded

the maximum germination percentage (66.7) and root length.

Soaking in 100 ppm IBA improved the shoot and root lengths and dry weight of

Syzygium cuminii better than other treatments (Vanangamudi et al., 1999). Naidu et al.,

(2000) studied the effect of growth regulators like GA3, IBA and IAA, 250 to 1500 ppm of

the above solutions were found to be effective in inducing Sapindus trifoliatus seed

germination. Increase in the duration of soaking period found to increase seed

germination. GA3 was found to be more effective in improving the seed germination in

soapnut (Sapindus emarginatus) than either IBA or IAA (Naidu et al., 2000). Seeds of

Strychnos nux-vomica soaking in 500 ppm GA3 for 24 hours then incubation of seeds at

40ºC for 3 days and alternate water soaking (16 hours) and drying (8 hours) for 14 days

significantly increased the germination compared to the control (Sivakumar et al., 2004).

Anoop et al., (2009) reported that the seeds of Pyracantha crinulata were recorded 94%

germination after 12 hours inhibition. Seeds of Punica granatum treated under 25-30 days

stratification recorded 91.66% maximum germination (Rawat et al., 2010).

Seeds of different plants contain different qualities of vitamins. The concentration

of these vitamins in seed of plants from one and the same variety grown under different

conditions also differ. Therefore, it is understandable that under certain conditions seeds

accumulate the quality of vitamins necessary for their germination, while under other

conditions, the vitamin content is insufficient. In prolonged storage, the vitamin content of

seeds gets decreased. Studies on the effect of vitamins on seed germination appear quite

promising. The role played by vitamins in the process of seed germination was noted by

17

Funk (1922). The treatment of rice seeds with 0.01 percent solution of ascorbic acid led to

an intensive synthesis of auxins (Skrabka, 1965). Glutamin (1967) reported that yield of

peas was increased when the seeds were pre soaked in 0.1 percent of ascorbic acid

solution. Dave and Gaur (1970) observed the stimulation of the vegetative growth,

enhancement of grain yield up to 30 percent, earlier ripening and heavier production in

barley due to the pre-soaking treatment of seeds with ascorbic acid. Viswanath et al.,

(1972) found that pre-soaking treatment of seeds of ragi with ascorbic acid showed better

germination capacity and more vigorous growth than untreated seeds. The beneficial effect

of ascorbic acid, pyridoxine and thiamine on root formation of Justicia gendarussa and

Ixora singaporensis were recorded (Mitra et al., 1984).

Huang (1988) studied the effect of ascorbic acid on germination and early growth

of Sesbania sesban. Seeds were pretreated with ascorbic acid solution at different

concentrations for 24 hours before germination. Both shoot and root lengths were found to

be significantly greater at 250, 500 and 1000 ppm concentrations. Seeds of Carica papaya

treated with ascorbic acid for 6 hours improved the vigour and yield (Ghanta et al., 1992).

The height of Eucalyptus tereticornis seedling was increased by the application of

nutrients such as nitrogen, sulphur, potassium and calcium (Hussian and Theagarajan

1966). Heydecker (1972) reported that potassium nitrate and thiourea had enhanced the

germination of Albizia lebbeck. Both pottasium nitrate and thiourea exhibited the highest

effect on germination rate i.e., total germination in Atropa belladona and caused better

seedling vigour (Choudhury and Kaul, 1974). Gosh et al., (1974) reported that Pinus

patula seeds soaked in 3% hydrogen peroxide (H2O2) for 6 hours displayed increased

germination percent. But in Pinus caribaea seeds, the response was good with H2O2

18

soaking (6%) for 6-18 hours. According to Roy (1985) when the seeds of Albizia lebbeck

were soaked in aqueous solution of pottasium nitrate and thiourea for 12 hours

germination percentage was enhanced. Seeds of Cyanodon dactylon treated with 10%

ethanol increased the germination (Salchi et al., 2008). Seeds of Solanum melongena

healed with country made liquor showed 70% maximum germination (Sanjeev Sharma

and Kapil Sharma, 2010).

Msanga and Maghembe (1986) observed low percentage of seed germination

ranging from 10-33.5% after 36 days when the seeds of Albizia schimperana were treated

with KNO3, H2O2 and ethanol. Treatment with KNO3 had been reported to increase

germination in loblolly pine (Biswas et al., 1972) and in Citrus limonis (Choudri and

Chakrawar, 1980). KNO3 had reported to raise the ambient oxygen level by making less

oxygen available for the citric acid cycle (Bewley and Black, 1982). One percent

pottasium dihydrogen phosphate enchanced the germination percentage up to 88 percent in

Zizyphus mauritiana (Ghosh and Sen, 1988). Soaked seeds of Ephedra foliata in aqueous

solutions of different concentrations (10-100 ppm) of KNO3, KH2PO4 or MgSO4 for 24 hours

promoted germination compared with the water soaked control (Shashikala et al., 1989).

Nagaveni et al., (1989) recommended soaking of sandal seeds in H2O2 (1%) or

SC(NH2)2 (5%) or methanol extract for obtaining uniform seed germination. Another

significant advantage of soaking in the above mentioned chemicals was that the soaking

period could be shortened. KNO3 and NaHClO3 enhanced the germination percentage up

to 61 and 74.41% (Jitendra Butola and Hemant Badola, 2004).

Santosh Kumar et al., (2007) reported that the Pongamia pinnata seeds treated

with hot water for 30 minutes recorded germination percentage up to 100 percent.

19

Sinhababu et al., (2007) reported that Cassia fistula seeds were treated with 80°C hot

water for 5 minutes increased the germination up to 100%. Bauhinia thonningii seeds

were treated with hot water for 5 minutes increased the germination up to 53.3% (Weston

Mwase and Thokozile Mvula, 2011). According to Zahra Karimian Fariman et al., (2011)

seeds of Echinacea purpurea treated with cold water for 24 hours showed 98%

germination.

Kajamaideen et al., (1990) reported that the Casuarina equisetifolia seeds treated

with 1.5% KNO3 and 7.5% calcium oxychloride (CaOCl2) for 36 hours recorded

germination up to 65 and 63 percent, respectively. These treatments also increased the

vigour index. Seeds of Peltophorum ferrugenium soaked with 1% KNO3 and 0.1%

SC(NH2)2 recorded enhanced precentage of germination (Mukhopadhyay et al., 1990).

Ramakrishna et al., (1990) reported significant increase in germination and vigour index

of Ailanthus excelsa seeds treated with 2.5% KNO3 over cytozyme, mixtalol, calcium

oxychloride and control. Soaking of sorghum seeds in chemical solutions like KNO3 and

KH2Po4 increased the seedling emergence, plant height, dry matter production and vigour

(Periathambi, 1980; Vanangamudi and Kulandaivelu, 1989 and Kajamaideen et al., 1990).

According to Roy (1992b), seeds of Albizia lebbeck treated with 0.3% KNO3 and 0.2%

and 0.3% SC(NH2)2 gave increased germination of 69.3, 75.6 and 73.6 percent,

respectively. Soaking of neem seeds possessing 12 percent moisture in 2% KNO3 for 24 hours

followed by a few hours of air drying enhanced seed germination (Vanangamudi, 1993).

Nayital et al., (1993) reported that surface sterilizing of western larch seeds with

0.75% sodium hypochlorite for 5 minutes significantly increased the germination

percentage. Studies on seed fortification with different chemicals in neem revealed

20

enhanced germination as a result of treatment with 2% KNO3 compared to water treatment

(Ponnusamy, 1993). Ching and Lin (1994) reported that soaking fresh Cinnamomum

camphora seeds in 15% hydrogen peroxide for 25 minutes significantly increased the

germination percent over the control. Scarified seeds of Acacia nilotica soaked in 2.0%

potassium nitrate for 24 hours recorded significant increase in germination percentage and

biomass over control and other treatments (Palani et al., 1995). Seeds of Foeniculum

vulgare soaked in 100ppm Salicylic acid for 12 hours increase in germination up to 80%

(Asghar Farcyollahi et al., 2010).

Bisht and Kediyal (1995) reported that Atropa belladona seeds treated with 75%

H2So4 for 2 minutes followed by 30% Sodium hydroxide (NaOH) for 5 minutes showed

good germination (96.5 percent) and survival of seedlings. Kumaran et al., (1996) treated

seeds of Azadirachta indica with potassium chloride, potassium dihydrogen phosphate,

KCl, KH2Po4, KNO3 and (NH4)2HPO4 each at two concentrations of 1% and 2% for a

duration of 2 hours and observed that soaking in 2% KH2Po4 gave maximum germination,

shoot length and number of leaves and 1% KH2Po4 gave maximum root length.

Soaking the seeds of red sanders (Pterocarpus santalinus) in ammonium nitrate,

potassium nitrate and thiourea solutions ranging from 100 to 500 ppm was found to be

effective in inducing germination. Generally 400 ppm concentration of all the aforesaid

chemicals resulted in maximum percentage of seed germination (Naidu and

Rajendrulu, 2001). Azhdari et al., (2010) reported the seeds of Medicago sativa treated

with KCl was improve the germination up to 96%.

Seed moisture is the most important factor affecting the retention of viability in

storage. When freshly harvested recalcitrant seeds are dried, viability is at first slightly

21

reduced as moisture is lost, but then begins to reduce considerably at a moisture content, termed

as “critical moisture content” (Tompsett, 1984). Seeds of Artocarpus heterophyllus stored at

20ºC retained viability for 5 weeks with 41% germination (Rekha Warrier et al., 2009).

The optimum range of moisture content for many of the large hardwood seeds is

25-79% (Wang, 1974). The exact causes of recalcitrant seed death and its relationship

with moisture content are not fully understood (Fu et al., 1993). Chin et al., (1989) stated

that loss of viability could be either due to the moisture content falling below a certain

critical value or simply a general physiological deterioration with time.

Critical moisture content varies greatly among the recalcitrant species, cultivars

and seed lots (Chin, 1988; King and Roberts, 1979) and depending upon the stage of seed

maturity at time of collection (Finch-Savage and Blake, 1994; Hong and Ellis, 1990).

Most of the recalcitrant seeds did not survive desiccation from 25 to 15% while no seed

survived desiccation below 10% moisture content (Ellis, 1991). The critical moisture content

may also vary with the method of seed drying (Farrant et al., 1985; Pritchard, 1991). The values

of the “lowest safe moisture content” vary between extremes of about 23 percent for

Cocoa (Theobroma cocao) (Mumford and Brett, 1982) to 61.5 percent for

Avicennia marina (Farrant et al., 1986). Despite this variation, these moisture contents are

equivalent to a relatively narrow band of relative humidities of 96 to 98 percent, or seed water

potentials of about 1.5 MPa to –5MPa (Poulsen and Eroiksen, 1992; Pritchard, 1991).

The storage temperature and viability are negatively correlated. With reduction in

temperature, the respiration rate is reduced and the viability is increased (Harrington,

1963). Harrington (1970) suggested that between 50°C and 0°C, every 5°C reduction in

storage temperature doubles the storage life of seeds. The viability of Syzygium cuminii

22

seeds was observed to be 3 months (DFSC, 1999) to 5 months (Mittal et al., 1999a). Sunil

Chandra Joshi and Pant (2010) noticed the seeds of Salvia sclarea stored at 25°C recorded

maximum germination (42%) compared to room temperature (40%). Chilling temperatures

are known to adversely affect the viability of seeds of tropical origin (Chin et al., 1989;

Bedi and Basra, 1993). Chilling injury occurs at between 5 and 10°C for

Shorea roxburghii, 5 and 12°C for Symphonia globulifera and Hopea odorata (Corbineau

and Come, 1988).

Seeds of Myristica fragrans are also desiccation sensitive below 31.05%, but the seeds

are able to tolerate up to 5°C (Sangakkara, 1993). Storage temperatures below 15°C were

reported to be lethal for most of the tropical recalcitrant seeds (Bedi and Basra, 1993). Tang

and Tamari (1973) stated that a period at 15°C had conditioned Hopea helferi and Hopea

odorata seeds, so that they could tolerate 10°C on transfer after 5 weeks.

In tropical climatic conditions storage practices requiring high moisture often

provide ideal conditions for mould invasion, proliferation and elaboration of mycotoxins

(Singh and Khan, 2001). Manifestation of effects of fungi on seeds depends upon

susceptibility of seeds to fungi, pathogen, virulence, amount of inoculum present n seeds

and environmental factors (Shea, 1960). High seed moisture content also increases fungal

activity. Attack of mould fungi is a constraint limiting seed germination (Singh et al., 1979).

Apart from depreciating the quality of the seeds, fungi also cause decay of seeds during

germination (Aswatanarayana et al., 1996). The problem of storing recalcitrant seeds in

good condition is exacerbated by action of fungi (Berjak et al., 1990). The natural loss of

seed viability, which is accompanied with leakage of biomolecules due to loss of

23

membrane integrity, in conjunction with relatively warm humid storage environment

provides and excellent milieu for fungal proliferation (Mycock and Berjak, 1990).

The composition of mycoflora in recalcitrant seeds was found to narrow down

with increasing storage time (Mycock and Berjak, 1990), with Fusarium species often

becoming dominant. Single or multiple infections by these species are responsible for

quick deterioration and decay of the seeds. Attack by pathogens has been reported as a

serious cause for loss of viability in high moisture content seeds like Syzygium cuminii

(Mittal et al., 1999a). Twenty-four fungi belonging to 20 genera were identified on freshly

collected seeds. In Hevea brasiliensis seeds, 21 species of fungus have been reported in

Malaysia (Dalbir-Singh et al., 1990). The high incidence of different fungi on

Dipterocarpus retusus seeds was reported to be due to prevailing ambient tropical temperature

and relative humidity, which encourage the growth and development of these fungi

(Mohanan and Sharma, 1991).

Seed pathogens are playing dominant and crucial role not only as seed

deteriorating agents, but also as primary and secondary incidents of the seedling diseases.

By adopting suitable prophylactic measures starting from seed collection to seed sowing,

the influence of mycoflora can be avoided. The need for protecting the moist

dipterocarp seeds against fungal attack was felt by Jansen (1971), who stated that seeds of

Neobalanocarpus heimii have increased storage life when treated with fungicides.

Fungicide treatment is probably the cheapest and often the safest method of control

of seed-borne fungi (Sharma, 1989). Benomyle and NaClO treatments were effective in

controlling the mycoflora during storage of Syzygium cuminii seeds (Mittal et al., 1999a).

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Seed treatment with sodium hypochlorite reduced the occurrence of fast growing

Aspergillus niger and Penicillium species in Eugenia dysenterica (Mittal et al., 1999b).

On the other hand, the longevity of Hopea helferi seeds was reduced by fungicide

treatment (Tang and Tamari, 1973).

In many agricultural crops biocides are used to control seed borne fungi. Spraying

leaf extracts of Delonix regia, Pongamia glabra and Acacia nilotica during flowering

reduced the seed borne pathogens viz., Alternaria helianthi, Macrophomina phaseolina

and Rhizoctonia solani in sunflower (Thiribhuvanamala, 1996). Treatment of sorghum

seeds with leaf extract of neem effectively controlled grain mold (Lakshmanan et al.,

1988). Leaf extract of Ocimum occidentalis and Vernonia amygdaline is effectively

reduced seed borne fungal pathogens of mungbean seeds (Onuegbu, 1999).

Recent studies have shown that vapour extracted essential oils of many different

tropical and temperate plant species are highly potent biocides that in very low

concentrations can control or kill fungi or bacteria by at least the same efficiency as

synthetic anti-microbial compounds and as such they can be applied as surface

disinfectants or fumigants (Paster et al., 1995).

There is no satisfactory method of maintaining the viability of recalcitrant seeds

over the medium and long term. They cannot be dried and stored at sub-zero temperatures,

because they would be then killed by freezing injury resulting from ice formation.

The longevity of recalcitrant seeds is short, from a few weeks to a few months for species

adapted to tropical environments (King and Roberts, 1979), and up to about 3 years for

several species adapted to temperate environments (Suszka and Tylkowski, 1980).

However, if the optimum storage environments are carefully determined, longevity of

25

several tropical recalcitrant seeds can be extended to 3 years, as that to Symphonia

globulifera (Corbineau and Come, 1988).

The principle of successful moist seed storage for recalcitrant seeds is that seeds

must be maintained at moisture contents close to that at which they are shed, with

continuous access to oxygen; these circumstances minimize seed deterioration, since

repair mechanisms can operate (Villiers, 1972). Under these conditions (high seed

moisture and available oxygen), however, seeds tend to germinate. It is also essential that

the conditions should prevent or at least delay germination. It is therefore, easier to store

recalcitrant species with dormant seeds (either primary dormancy or induced dormancy)

than with nondormant seeds under such conditions. For nondormant seeds, as shown by

most tropical tree seeds at maturity (King and Roberts, 1979), low temperatures can

reduce the rates of both seed deterioration and germination provided they remain above

the value that results in chilling damage or the lower value at which ice crystallization

occurs. Sivakumar et al., (2006) suggested that the seeds of Strychnos nux-vomica stored

at ambient temperature for 30 weeks showed highest germination (92%).

Furthermore, seeds of many temperate species with recalcitrant seed storage

behaviour have the ability to germinate at 2 to 5°C, and thus, moist storage of such

recalcitrant seeds at 0 to 10 °C may result in germination during storage. In such cases,

germination may be prevented by reducing seed moisture content slightly (by about 5 per

cent below that of fresh seeds), or either reducing the storage temperature to below the

optimum prechilling temperature (eg. 0 to -3°C) (Suszka, 1978) or increasing it to the base

temperature for germination of dormant seeds (Pritchard et al., 1996). The viability of

seeds of sycamore (Acer pseudoplatanus) and Quercus robur can be maintained for

26

3 years at 24 to 32 percent and 40 to 45% moisture content, respectively, at –1 to –3°C

(Suszka, 1978), and horse chestnut (Aesculus hippocastanum) for 3 years, moist dormant

seeds are stored at 16°C, although in this case subsequent prechilling is required to remove

dormancy (Pritchard et al., 1996). Obembe and Agboola, (2008), recorded seeds of

Occimum gratissimum stored at 25º C for 12 hours under light condition released the

dormancy and ready to germinate.

The optimum storage temperature varies from about 7°C to 17°C among species

adapted to tropical climates, and between about -3°C and 5°C among many of those

adapted to temperate climates (Hong et al., 1996). The optimum temperatures appears to

be that at which non-dormant seeds remain alive but are unable to germinate, i.e. the base

temperature for germination (Corbineau and Come, 1988). The optimum storage

temperatures determined in this way are 10°C for Shorea roxburghii, 12°C for

Mangifera indica and15°C for Hopea odorata and Symphonia globulifera (Corbineau and

Come, 1988). The seeds of Ruellia species stored at 25°C is the optimum for germination

(Carlos Cervera and Victro Parra-Table, 2009). However, recalcitrant seeds of species

adapted to temperate climates show considerable dormancy, which requires long periods

of prechilling at 2 to 5°C to overcome.

Maintaining recalcitrant seeds at high moisture content, with continuous aeration,

and at the same time preventing germination and fungal contamination, is difficult.

Aeration can result in loss of seed moisture, and respiration can quickly deplete oxygen.

The storage medium is therefore, very important for recalcitrant seeds. It should fulfill two

functions: first, to maintain seed moisture content at high values and second to allow

diffusion of sufficient oxygen to the moist seeds. The storage of moist recalcitrant seeds in

27

damp charcoal, sawdust or moist sand is generally reported to be more efficient than

storage in polyethylene bags. Storage at or near the harvest moisture content in media such

as sawdust (at 16% moisture content) or perlite (at 0 to 4% moisture content) within

suitable containers, such as open weave sacks or bags, placed in a high humidity room has

been recommended (Tompsett and Kemp, 1996).

Schaefer (1991) stored seeds of Podocarpus milajianus and Prunus africana in

cold moist sawdust, which also helped to reduce fungal infection. Storage in media with

some moisture-retention capacity to prevent desiccation has been found suitable for some

species. Song et al., (1986) stored seeds of Hopea hainanensis in moist coconut dust, and

perlite has been used successfully for a number or recalcitrant species. Application of

natural germination inhibitors like abscisic acid to prevent germination in storage has

largely failed to prolong viability (King and Roberts, 1979). Storage of recalcitrant Prunus

africana seed within the pulp, in which germination inhibitors occur, reduced viability of

seeds significantly as compared to extracted seeds (Schaefer, 1990). Using moist sand as

storage medium, germination of the Illicium verum seeds stored for two months was 70-75

percent and germination ability of seeds stored for 7 months was totally lost. The seeds

could be stored at sand to seed ratio of 2:1 under soil surface and storage could lose for

duration of 75-100 days (DFSC, 1999). Seeds of Nardostachys jatamansi stored at room

temperature exhibit viability of less than a year, whereas 0-4°C temperature storage

enhanced it more than two times (Chauhan and Nautiyal, 2007).

Seed storage in suitable containers would prevent the direct contact of seeds with

storage environment and also protect from pests and diseases. Hence for effective storage,

appropriate storage container is essential (Purohit and Doijode, 1988). Doijode (1995)

28

suggests that undried seeds of recalcitrant species are to be packed in semi-moisture

resistant containers and stored at medium temperature (10-15°C). Polyethylene bags with

a wall thickness from 0.1 mm to 0.250 mm are thick enough to prevent excessive moisture

loss and thin enough to allow some gas exchange for short-term storage of recalcitrant

seeds (Bonner and Vozzo, 1987; Bonner, 1996). Mittal et al., (1999a) documented that

Syzygium cuminii seeds with 43.6% moisture content could be successfully stored up to 20

weeks in untied polyethylene at 16 and 5°C without serious impact on their germination.

Polyethylene bags are suitable provided the material is thin and permeable enough to

permit some gaseous exchange (King and Roberts, 1979). A wall thickness of 0.1-0.25 mm

was found suitable to prevent excessive moisture loss, yet allowing some ventilation for

short-term storage of recalcitrant seeds (Bonner, 1996). Panochit et al., (1984) found that

seeds of Shorea siamensis stored at ambient temperature in folded plastic bags prolonged

viability as compared to those in sealed plastic bags, presumably because the sealing

reduced ventilation.

Seeds of Myristica fragrans were able to store at 5°C in sealed polyethylene bags

for a period of one month (Sangakkara, 1993). Recalcitrant seeds respire actively and will

suffocate in closed containers. They must be stored in relatively thin covers, which allow

plenty of air exchange. It is therefore, difficult to maintain the moisture content of the

seeds and water should be added frequently by spraying and mixing the seeds

(Thomsen and Stubsgaard, 1998).