Int. J. Life. Sci. Scienti. Res., 4(3): 1774-1779, May 2018

Proliferation of Shoot from First Leaf of Carthamus tinctorius L. (Safflower)

Sneha Mendhe¹*, Sana Sheikh²

¹Assistant Professor, Department of Botany, Dr. D.Y. Patil A C S Women’s College, Pimpri-18, Pune, India

²Assistant Professor, Department of Zoology, Dr. D.Y. Patil A C S Women’s College, Pimpri-18, Pune, India

*Address for Correspondence –Mrs. Sneha Mendhe, Assistant Prof., Department of Botany, Dr. D. Y. Patil A C S Women’s College, Pimpri-18, Savitribai Phule Pune University, Pune, India.

ABSTRACT- Callus induction and in vitro plantlet regeneration system for safflower (Carthamus tinctorius L.) using first leaf were optimized by studying the influence on organogenesis of seedling age, media factor, growth regulator and excision orientation. Supplementation medium with auxin and cytokinin ratio >1 enhance growth rate of callus culture. Growth regulators IAA, NAA, BAP, kinetin in the medium where found effective for callus induction and regeneration in all explants. The BAP 5mg/l, NAA 1mg/l, 5-7 explants and callus derived from cotyledonary. Explants were cut from basal region of cotyledon of 5-7 days old seedlings. As compared to the standard media SH-M and B-5 growth was superior in MS medium. Capitula induction was observed in callus mediated shoot from cotyledon and with sucrose, IAA, NAA and BAP. The well-developed plantlet was transferred to the field.

Key words- Carthamus tinctorius L., Safflower, Callus, MS medium, BAP, NAA

INTRODUCTION- Carthamus tinctorius L. (Safflower) Asteraceae is an important oil seed crop of semiarid subtropical regions of average temperature 17 - 20° C which appear to be best for vegetative growth and optimum temperature of flowering is 24 to 32°C. Due to high content of Linoleic acid it occupies unique position among oil seed plants ^[1]. The young plant is used as a leafy vegetable; seed oil is used for industrial and edible purpose ^[1,2]. Safflower is considered as salt tolerant specially sodium salt. Flower yield and pigment content of flower have gain economic importance ^[3,4] to increasing countries and their use in medicine for curing several diseases.

In vitro plant regeneration system is basic necessity for such approaches. Direct somatic embryogenesis from cotyledon explants ^[4] and in vitro shoot regeneration has been reported in safflower ^[4,5]. Modern techniques like embryo rescue and other biotechnological tool may play an important role in overcoming such barriers. Development of cytoplasmic-genetic male sterility, system for hybrid breeding, a successful outcome of ongoing efforts to use polyembryony for varietal improvement and confirmation of apomixes in safflower ^[6].

Genetic transformation of safflower to impart resistance to biotic and abiotic factor in addition to development of seed with altered fatty acid and protein profiles ^[7]. However cultivar can vary responses and regeneration of whole plant.

MATERIALS AND METHODS- Certified seeds of safflower (Carthamus tinctorius L.) were obtained on August 2011 from Department of Botany, National Environment Engineering Research Institute (NEERI) Nagpur, India. Seeds were surface sterilized with 0.1 % (w/v) mercuric chloride for (HgCl₂₎ with 3 minute constant shaking followed by three washes for 1 minute each in sterilized distilled water. Seeds were then germinated and grown on sucrose 3%, agar 0.8 % under photo period of fluorescent light. Explants (cotyledon) were isolated- 15 - 17 mm² from 5 to 7 days old seedlings. Medium was supplemented with BAP and NAA (500 μl BAP and 1250μl NAA were added and volume was made up 250 ml by adding distilled water). Then explants were transferred onto callus induction medium.

Induction and Callus- Callus induction was carried out on MS medium supplemented with BAP and NAA   in combination. After 21 days of inoculation completely differentiated dense mass of callus showing further regeneration ability was observe. After three weeks of culture, the responded explants where further transferred on fresh medium containing same concentration of BAP and NAA ^[5]. Each regeneration step was further carried out for period of 21 days subculture onto fresh optimum callus induction ^[8]. Shoot induction from explants and calli (250 mg- 300mg/culture) was carried out on MS containing BAP 5mg/L and NAA 3 mg/L. Regenerated shoot were about 1cm and were separated from explants and callus. Rooting of resulting shoots (1 - 1.5 cm long) from explants and callus was attempted on MS without growth regulator and with sucrose 1 - 9%, NAA 5mg/L, BAP 0.25 mg/L (in combination).

Hardening- Rooted plantlets were removed from culture vials after agar had been removed by washing with sterile water, the plantlets were planted in a pots containing 1:1 sterilized potting mixture soil and washed sand (with pebble size of 0.5 - 1.0 mm) ^[8]. The plants were placed outside in the shade (light max 83.46 m-2 s-1μm, temperature 25 +­/- 4°C) irrigated at 3 days interval with tap water ^[9].

RESULT- We select 3-7 day old seedling and first leaf as explant (Fig. 1). Most cotyledonary leaves elongated and formed green yellow coloured compact callus about 18-21 days after culture initiation (Fig. 2). Induction of callus was observed in all media there was no statistically difference among concentration of BAP and NAA (Table 1). After 22-23 days subculture (Fig. 3) and proliferation of shoot from first leaf was visible after 28-32 days in all media tested (Fig. 4) shoot primordia developed into normal shoot after 40-43 days after culture initiation all concentration of BAP and NAA (Fig. 5 and Table 2).

Regeneration response was best on the MS medium supplemented with 1mg/lit NAA & 5mg/lit BAP. Callus induction was observed by using in first leaf explants and direct shoot regeneration was observed. Brownish green slow growing friable callus was obtained after 18 days of inoculation & shoot regeneration was obtained after 32 days of inoculation.

Table 1: Observation for Callus induction

Type of explants	Medium	Additional components in medium	Duration
			7	14	21	28
First leaf	MS	NAA- 3mg/lit BAP-5mg/lit	S	+	++	+++

Swelling= S, Callus induction= +, Callus Induction and Growth=  ++,  Callus Induction growth with good response= +++

Table 2: Observation for Shoot regeneration from callus

Type of explants	Medium	Additional components in medium	Duration
			7	14	21		28
First leaf calli	MS	NAA- 1mg/lit BAP-5mg/lit	+	S		S	S

Shooting= S, Primary response=  +        

DISCUSSION- According to ^[2] cultered 10 day old seedling with different concentration of TBZ and IAA induced shoot regeneration from cotyledonary leaves and In Vitro multiplication in safflower. Most published report ^[7] describe use of TBZ and IAA  But we are using only BAP and NAA the best shoot multiplication was achieved in range of media supplimented with BAP 5mg/lit and NAA 1mg/lit. Without using TBZ and IAA we got good result in BAP and IAA.                                                   

The concentration of hormones were changed and their growth were studied. For callus induction, different combination of BAP and NAA were taken if we take: BAP 5mg/L; NAA  5 mg/L, BAP 0.2 mg/L; NAA 5 mg/L, BAP 3 mg/L; NAA 5 mg/L then no response was seen. But in BAP 5 mg/L; NAA 3 mg/L combination best growth was observed. For shoot induction  in combination  BAP 3 mg/L; NAA 0.5 mg/L  no response was seen but in BAP 5 mg/L; NAA 1 mg/L growth was observed. We are using different concentration but explant were very favourable explant with high multiplication ratio 100% at concentration of BAP 5mg/lit and NAA 1mg/lit. All regenerated shoot tip (15-20 mm length) were excised and rooted radially in half strength of MS medium supplimented. Rooting was observed from cut end of shoots within 40-42 days in most media tested. All developing roots were physically vigorous and healthy (Fig. 6). 

CONCLUSIONS- We are concluded that proliferation of Carthamus tinctorius (Safflower) was effectively influenced by sucrose as a carbon source and hormone BAP and NAA. The optimum concentration of carbon source was found to be approximately 8% which is 8 g/lit by good response of first leaf cutting. The effective concentration for induction of callus was found to be BAP 5 mg/lit & NAA 3 mg/lit and BAP 5 mg/lit and NAA 1 mg/lit for shoot induction. Carthamus tinctorius (Safflower) was taken as a study of interest as it has many medicinal values so as to make people aware of it. We can produce genetically modified crop of Carthamus tinctorius to make it resistance to biotic and abiotic factor. We can use the stigma of Carthamus tinctorius as alternative to saffron as it is costly.

ABBRIVATION- IAA: Indol Acedic acid, IBA: Indol butyric acid, NAA: Alpha naphthalene acetic acid, MS: Murashige and Skoog, B5: Gamborg, SH - M: Micchell and Gildow.

ACKNOWLEDGEMENTS- We express our thanks and gratitude to P.G.T.D of Botany RTM Nagpur University, Prof. Dr. T Srinivasu, Mrs. Madhuri Thakre for their precious guidance and providing laboratory facilities and a special thanks to Dr. Ranjit Patil (Principal of   Dr. D.Y. Patil ACS Women’s College, Pimpri-18)  for his constant support.

CONTRIBUTION OF AUTHORS- The work was designed and performed by Mrs. Sneha Mendhe along with collection of materials and data collection. Ms. Sana Sheikh was analyzed the data and interpreted the work.

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