Volume 19(1), 1- 6, 2015 JOURNAL of Horticulture, Forestry and Biotechnology

www.journal-hfb.usab-tm.ro

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The Evaluation Effects of Some Vegetative Propagation Methods and Plant Growth Regulators on Bulblet Production Rate in Crown Imperial (Fritillaria imperialis L.) Mousa Solgi 1*, Kheirola Dastyari 2 and Ebrahim Hadavi2

1Department of Horticultural Sciences, Faculty of Agriculture and Natural Resources, Arak University, Arak

38156-8-8349, Iran, 2Department of Horticulture, Karaj Branch, Islamic Azad University, Karaj, Iran.

*Corresponding author’s email: M-solgi@araku.ac.ir Abstract This study was conducted to evaluate the effects of different vegetative propagation methods and some PGRs on adventitious bulblet production of Fritillaria imperialis in Iran for the first time. Mother bulbs were soaked in a solution containing equal doses of NAA plus BA in four levels (0, 100, 200 and 300 mg/L) and four vegetative propagation methods (i.e. scoring, chipping with 4-chip, chipping with 8-chip and horizontal). Then after, they sealed in plastic bags containing equal volumetric proportions of humid vermiculate and perlite. Results showed that, there is a significant difference between various vegetative propagation methods and levels of PGRs (NAA plus BA). The highest average number of bulblets production per bulb was achieved from 8-chip method and the least average number was from scoring method. Treating bulbs by using 300 mg/L NAA + 300 mg/L BA indicated maximum bulblet regeneration while the least ones were observed in no PGRs treatment. In our experiment, the application of 300 mg/L NAA plus 300 mg/L BA in combination with horizontal cutting of bulbs yielded the highest bulblet formation per mother bulb (31.3) which was significantly higher than control treatment with five bulblet per bulb. We conclude that the application of proper vegetative propagation method combined with the use of NAA plus BA could be a reasonable outcome in term of propagation rate.

Key words Crown imperials,Vegetative propagation, Chipping, Bulb, Bulblet, PGRs, BA, NAA.

Fritillaria imperialis L. belonging to the Liliaceae

family has a high ornamental and medicinal value.

Because of big and attractive bell-shape florets, it has

been used as cut flower, pot plant and in landscape

design and has revealed a great commercial potential

(1). Its bulb is an important source of anti-tussive,

expectorant and anti-hypertensive drugs and has high

starch content so can be considered as new starch

source for the food and medicine industry (2) and is an

important source of steroidal alkaloids which have

cholinesterase inhibiting role (3). However in Iran, in

addition to the above advantages, it is more important

for its role in natural scenery, as numerous plains of

eye-catching wild crown imperial. Every year in early

May to mid-June slopes and highlands of Zagros

mountains will be covered by red and yellow flowers

and attracts many tourist. Therefore, its ecotourism

aspects must be significantly concerned.

Iran is origin and center of genetic diversity of

F.imperialis. Unfortunately, the wild populations in

recent years are at risk of extinction due to habitat

destruction, eradication of the plants with their bulbs

and transfer them to florists' shops by native people,

digging bulbs by tourists or native people for planting

bulbs in the yard and gardens, pests overflow, irregular

grazing of Fritillaria stands, changing of pastures to

dry farms and lack of protecting rules (4). Not only its

vegetative reproduction rate in nature is slow (max. 2-4

bulblet per bulb upon climate conditions) but also the

reproduction cycle from seed to develop plant capable

of flower production takes 5-7 years (5). Therefore, its

destruction rate is much higher than the natural

replacement rate. To prevent the destruction of their

natural habitat, an effective, simple and available

propagation protocol for local farmers is necessary.

This, in addition to reducing pressure on wild

populations of plants, creates a new revenue source for

local farmers (6).

Based on Van Leeuwen et al. (1997), Mori et al.

(1997), Yanagawa (2005) and Ulug et al. (2010),

application of some vegetative propagation methods on

some geophytes' bulbs can leads to producing more

bulblet than that in nature (7, 8, 9, 10). Vegetative

propagation has various techniques such as chipping,

scaling and cross-cutting (including various

applications such as: scoring and horizontal cutting).

Chipping is cutting a bulb longitudinally into number

of segments (chips) in equal size. By this action the

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main growing point on basal plate is destroyed and

apical dominance is broken (7). In this method, loss of

water encourages production of bulblet and leaving

bulb pieces in dry sand for a week lead to callus

formation. After formation of callus, segments put in

plastic bags containing wet vermiculite or perlite and

left in dark place at 20-30 °C for two months for

growth of bulblet. Also, the application of hormones

(11) in combination with vegetative propagation

methods may be effective in increasing the bulblet

production (11). As F. imperialis bulb has low number

of scales (3-5 scales); therefore, application of cross-

cutting or chipping methods, which provide more

surface of cutting in each bulb together with fast

reproduction period, was carried out in combination

with some concentrations of PGRs in this study.

Material and Methods

1- Preparation of bulbs and medium

Wild mother bulbs were collected from their natural

habitat (at 1400 m height from sea level in Zagros

Mountains, located in Ilam province, Dareshahr city)

and were washed to remove mud and all surface

contaminants. Outer decayed fibrous layers, dried roots

and wounded surface were removed with a scalpel, too.

Equal volumetric proportions of fine vermiculate and

perlite soaked in Thiophanate-Methyl, (70% WP)

fungicide solution (1 g/L) for 30 min, then sieved and

left in room temperature to reach proper wetness and

was used as propagation medium. All mother bulbs and

bulb segments (chips) was disinfected also. All

equipment such as knives, cutting boards, gloves,

containers and plastic bags were first soaked in 10% of

Sehat®, commercial sodium hypochlorite (Sehat Co,

Iran) containing 5% available chlorine for 30 min.

Then washed three times and disinfected in same

fungicide solution mentioned before.

2- Preparation of hormonal solutions and hormonal

treatments

Plant hormones BA and NAA were manufactured by

MERCK® company (Germany). Concentrations of 100,

200 and 300 mg/L of both BA and NAA hormones

were prepared. 1 N KOH and 1 N NaOH were used as

BA and NAA solvent, respectively. To prepare

solution (100+100), 100 mg of BA was dissolved in

100 ml of KOH, and 100 mg of NAA was dissolved in

100 ml of NaOH. Then, these two solutions combined

together and diluted to 1000 mL of volume in distilled

water to form a solution containing 100 mg/L of both

NAA and BA. To form 200+200 and 300+300

solutions, 200 and 300 mg of both hormones was

weighed as mentioned above, prepared, labeled and

stored in refrigerator in 5 °С for 2 days. Bulb groups

were soaked for 10 min in an appropriate solution of

the hormone levels (13).

3- Cutting the bulbs and drying bulb pieces

Mother bulbs were classified into four group (each of

them consisted of 24 bulbs) and were soaked in

different concentrations of PGRs solutions. All bulb

segments prior to putting in the bags, were left in dry

sand for one week to callus formation. For each group

of bulbs the following vegetative propagation

techniques were applied (10): Scoring: Lower half of

mother bulbs were divided into four equal parts by

cutting across the base of bulbs (one-fourth to one-third

of the height of the bulb in depth). In this way the

growing potential was collected at the bottom of the

bulb plate which encourages the formation of bulblets. Chipping (4-chip) :Mother bulbs from top to bottom

were vertically cut into four equal segments. A little

part of basal plate was left on each of the bulb

segments.

Chipping (8-chip): Mother bulbs from top to bottom

were vertically cut into eight equal segments. A little

part of basal plate was left on each of the bulb

segments.

Horizontal Cutting: Mother bulbs were cut horizontally

into two equal parts (from one side to the other, across).

The basal plate was in lower part.

4- Planting treatments and culture conditions

The propagules were mixed with humid medium and

put in black pastic bags. The bags were sealed leaving

air spaces inside and placed in dark (12) at relatively

cool temperatures (23.5 ± 3.5 for day and 17.5±2.5 °C

at night-time) for at least two months. The Bags were

checked two times for fungal infections and rot.

Decayed propagules were removed and healthy ones

were disinfected again with fungicide solution and re-

cultured in fresh medium.

5- Statistical method

The experiment was conducted as Factorial in a

completely randomized design consisting of two

factors. First factor was cutting methods in four levels

(scoring, chipping (4-chip), chipping (8-chip) and

horizontal cutting) and the second factor was hormone

concentrations (NAA plus BA) in 4 levels (0, 100, 200

and 300 mg/L) along with control. The number of

bulblets formation and their weights were recorded

after two months. Data were analyzed using the

ANOVA procedure of SPSS statistical software.

Duncan's multiple range test (DMRT) were performed

to determine the statistical significance of differences

between means of treatments at the 5% probability

level.

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Results and Discussions

1- The effect of vegetative propagation methods on

bulblet number and weight

Our findings showed that the effects of different

applied vegetative propagation methods were

significant on bulblet number formation and weight in

comparison with the control (p≤0.05) (Table 1). The

highest and the lowest average number of bulblet

formation per mother bulb produced by chipping (8-

chips) and scoring method, respectively (Table 1 and

Fig.1). The average number of bulblet with the

increasing number of chips increased while the average

weight of bulblet decreased (Fig. 5). The minimum and

maximum average weight of bulblet observed in

chipping (8-chips) and scoring method, respectively

(Table 1 and Fig. 2).

Table 1

Effect of vegetative propagation methods and PGRs on bulblet

number and weight of Fritillaria imperialis L.

Cutting type Concentrations

Of NAA+BA (mg/L) Average number*

of bulblet

Average weight*

of bulblet (g)

Scoring

0,0 5.0 e 0.95 abc

100,100 7.8 cde 0.35 cde

200,200 8.3 cde 0.89 abcd

300,300 13.0 bcde 0.71 abcde

Chipping (4-chip)

0,0 16.7 abcde 1.27 ab

100,100 20.0 abcde 1.03 ab

200,200 24.3 abc 0.69 abcde

300,300 23.0 abcd 0.56 bcde

Chipping (8-chip)

0,0 13.7 bcde 0.66 bcde

100,100 30.0 a 0.44 bcde

200,200 26.7 ab 0.56 bcde

300,300 20.0 abcde 0.27 e

Horizontal

0,0 10.0 cde 0.74 acde

100,100 22.3 abcd 0.47 bcde

200,200 21.7 abcd 0.54 bcde

300,300 31.3 a 0.30 de

*Means in the same column followed by the same letter are not significantly different by Duncan's multiple range test

(p≤0.05).

0

5

10

15

20

25

Scoring Chipping (4chip) Horizontal Chipping (8chip)

Vegetative propagation method

Bu

lble

t n

um

ber

aababc

d

Fig. 1. Effect of vegetative propagation method on bulblet average number per mother

bulb of Fritillaria imperialis

4

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

chipping (8 chip) Horizontal Scoring chipping (4 chip)

Vegetative propagation method

Bu

lnle

t w

eigh

t (g

)

a

ab

bcbcd

Fig. 2. Effect of vegetative propagation method on bulblet average weight per mother

bulb of Fritillaria imperialis

These results are in agreement with findings of

researchers approving that with increasing the number

of chips per mother bulbs, the number of bulblets

increase but the weight of bulblets decrease. For

instance Ulug et al. (2010) cut mother bulbs of

Fritillaria persica L. into segments by various

vegetative propagation techniques and reported that by

increasing number of chips per mother bulb, the

average number of bulblet per bulb increased and

conversely, the average weigh of bulblet per bulb

decreased (10). Seyidoglu and Zencirkiran (2008) and

Zhu et al. (2005) by application chipping method on

bulb of Sternbergia lutea and Hippeastrum hybridum

also reported that increasing in chips, increased the

number and decreased the weight of bulblets per

mother bulbs (6, 14).

2- The effect of PGRs concentrations on bulblet

number and weight.

Results showed that the effects of different

concentrations of NAA plus BA were significant on

bulblet number formation and weight in comparison

with the control (p≤0.05) (Table 1). The highest and

lowest average number of bulblets per mother bulb

produced by application of NAA plus BA (300 mg/L)

and without PGRs treatment, respectively (Table 1 and

Fig. 3).The average number of bulblets with increasing

the concentration of the hormones increased and the

average fresh weight of bulblets per mother bulbs

decreased.

0

5

10

15

20

25

NAA0+BA0 NAA100+BA100 NAA200+BA200 NAA300+BA300

Hormons concentration (mg/L)

Bu

lble

t n

um

ber

aababc

d

Fig. 3. Effect of PGRs on bulblet average number per mother bulb of

Fritillaria imperialis L.

5

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

NAA300+BA300 NAA100+BA100 NAA200+BA200 NAA0+BA0

Hormons concentration (mg/L)

Bu

lble

t w

eig

ht

(g)

a

ab

bc

bcd

Fig. 4. Effect of PGRs on bulblet average weight per mother bulb of

Fritillaria imperialis L.

The minimum and maximum average fresh weight of

bulblet observed in application of NAA plus BA (300

mg/L) and without PGRs treatment, respectively

(Table 1 and Fig. 4). These results are in agreement

with findings of Kumar et al. (2008) which treated

oriental lily hybrids root explants with some PGRs in

MS medium for examination their effect on bulblet

production and reported that using of NAA and BA

alone and in combination in different concentrations

showed various effects on bulblet number and fresh

weight (15).

B

A

D

C

Fig. 5. A; bulblet produced from scoring vegetative method, B; bulblet produced

from horizontal vegetative method, C; bulblet produced from chipping vegetative method,

D; new plant grown from new produced bulblet.

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They observed that when concentration of NAA

plus BA increased from 1.5 mg/L to 2 mg/L, the

average number of bulblets per explants increased but

the average fresh weight decreased. Although, the interaction effect between vegetative

propagation method and PGRs were not significant but

was more effective than vegetative propagation method

or PGRs factors alone. The treatment “Horizonal +

NAA300+BA300”, “8-chips + NAA100 + BA100” and

“8-chips + NAA200+BA200” treatments which

produce average 31.3 , 30 and 26.7 bulblet per mother

bulb, respectively is higher than that of no PGRs

treatment.

Conclusion

In our experiment using 300 mg/L of NAA plus 300

mg/L BA in combination with Horizontal cutting of

bulbs yielded the highest average bulblet formation per

mother bulb which was significantly higher than that of

Scoring without PGRs treatment. We conclude that the

application of proper vegetative propagation method

combined with NAA plus BA is a reasonable outcome

in term of propagation rate of Fritillaria imperialis.

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