INTRODUCTION:

Cowpea (Vigna unguiculata L. Walp) is a major food crop that serves as an important source of protein, providing more than half the plant protein for human diets for many countries in Asia, Africa and America. It is an important source of dietary protein and also contain rich in carbohydrate, lipid, fiber, minerals and vitamins^1,2. Cowpea is one of the essential crops for rural population diet; it is the less costly source of protein (25% content) for rural people in West Africa³. Cowpea is also well known as soil biofertilizer due to its ability to establish an efficient symbiosis with nitrogen fixing Rhizobium. It increases the soil fertility through its symbiotic potential.

The cowpea plants are often exposed to numerous biotic and abiotic stresses which lead to decrease in the productivity. Sources of resistance to some of the biotic problems can be found in cowpea germplasm.

However, cultivated cowpeas are highly susceptible to insect pests and diseases which can cause substantial grain yield losses⁴. Incorporation of specific genes for abiotic and biotic stress tolerance in cowpea is low and diseases interspecific hybridization with wild relatives were not successful because of the compatibility barrier. Transgenic technology is one veritable alternative that my help breeders overcome these challenges, especially when applied alongside with conventional breeding.

Optimised cost effective regeneration system is required to commercialize the transgenic plant. genetic improvement of cowpea regeneration system that will provide totipotent cells which foster the development of complete plants is required. Due to its recalcitrant nature, numerous protocols have been standardized for in vitro regeneration in cowpea using various explants^5-10. However, none of these reports described an efficient protocol for in vitro regeneration in cowpea because of the difficulty in reproducibility and very low regeneration frequency¹¹. Although vitro regeneration techniques were reported in cowpea, many of the factors that regulate plant regeneration are not fully understood.

It has been already reported that the organic elicitors such as hemoglobin, casein hydrolysate, yeast extract, and glutamine enhanced the regeneration of tissues¹². Some of the natural biostimulants like seaweeds potential activity in stimulating the growth of the plants in vivo, but have been underutilized so far¹³. Utilization of seaweed and its products in agriculture has been increased in the past two decades. Seaweed products enhanced the growth and yield of various crops, improve nutrient mobilization, and favour the development of roots and shoots through environmental friendly approach¹⁴. Seaweeds extracts are marketed as liquid fertilizers and biostimulants since they contain many growth regulators such as cytokinins¹⁵, auxins¹⁶, gibberellins, betaines, macronutrients such as Ca, K, P, and micronutrients like Fe, Cu, Zn, B, Mn, Co, and Mo¹³, necessary for the development and growth of plants¹⁷. These positive effects are attributable to the presence of growth hormones that occur naturally in seaweeds that also are present in seaweed extracts that regulate plant growth^18,19. Hence with the above said importance, we aimed to screen the efficiency of Sargassum polycystum seaweed to regenerate the cowpea plantlets using node explant at in vitro conditions.

MATERIALS AND METHODS:

Collection of seaweed sample:

The marine algal species Sargassum polycystum was collected from coastal areas of Rameshwaram, Tamilnadu. The collected seaweed was washed in the seawater to remove the impurities and sand particles adherent to it and shade dried for a month.

Seaweed extraction:

The collected seaweed was made into powder and extracted with distilled water. Seaweed extract was prepared in such the way that 100g of dried sample was added to 1000 ml of distilled water and boiled in water bath at 70 ̊ for 30 min. Then seaweed extract was filtered with Whatman filter paper. The filtrate obtained was prepared at different concentrations from 10% to 50% and stored in refrigerator for further use.

Inoculation of seed explants:

The cowpea seeds were soaked in water supplemented with 1 mg/L of GA₃in an orbital shaker for overnight. Then the seeds were surface sterilized at in vitro condition with 70% ethanol for 1 min and 0.1% Mercuric chloride for 30 seconds. The sterilized seeds were rinsed with sterile distilled for 5 times to remove the traces of mercuric chloride with the seeds. The surface sterilized seeds were inoculated in the modified MS medium supplemented with MS salts and B₅ vitamins containing different concentrations (10-50%) of seaweed extract. The inoculated cultures were incubated at dark condition for 24 hours and observed for germination. The each experiment was repeated thrice and data was recorded after 3 days.

Inoculation of shoot tip and Node explant:

From the in vitro germinated seeds, cotyledonary node explants was excised with the dissecting sterile blade. With the help of sterile forceps, note cotyledonary node explants were inoculated in the medium containing different concentrations of TDZ (0.5-2.5 mg/L), Kinetin (0.2-1 mg/L) and different concentrations of seaweed extracts (5-25%). The inoculated conical flasks were cotton plugged and maintained under cool white fluorescent lights in tissue culture laboratory and the experiment was performed thrice.

Shoot elongation:

The shootlets of about 2cm were sub cultured in the fresh medium supplemented with different concentrations of seaweed extracts (5-25%). pH of the medium was adjusted to 5.8 by adding 0.1N HCL or 0.1N NaOH prior to autoclaving. Then the medium was autoclaved at 121°C for 15 min at 15 lbs.

Inoculation of elongated shoots in rooting medium and hardening:

Elongated shoots was taken out from the conical flasks with sterile forceps and inoculated in the medium fortified with hormones such as NAA and IAA and seaweed extract in different concentration. The seaweeds were tested individually as well as in combination with commercially available PGR’s. Plants with well developed shoots and roots were removed from the conical flasks containing medium with sterile forceps. The removed plants were washed with running tap water to remove the gelling substance adherent to it. Then the plants were transferred to plastic cups containing sterile soil and maintained in the culture room condition for 3 days. Then the plants were transferred to room temperature to increase the survival rate of the tissue cultured raised plantlets.

RESULTS AND DISCUSSION:

Majority of population in India depend on low priced vegetarian food for meeting their dietary requirements. Pulse crops particularly, Cowpea is a valuable source of proteins, minerals and vitamins and further it also used as food, fodder, vegetable and green manure for crops. Genotypes with inherent resistance to biotic stresses are needed to increase the productivity of cowpea and make its production more profitable. Compared to other grain legumes Soybean and Common bean cowpea perform poorly under in vitro conditions. So we planned to develop simple regeneration protocol using plant growth regulators and naturally available seaweed extract Sargassum polycystum.

Seed germination:

Contamination free seeds were inoculated in the MS medium supplemented with 0-50% of Sargassum polycystum. Medium supplemented with 20% of seaweed extract shown 100% of germination after 3 days of culture. Medium supplemented with 50% of seaweed extract shown least percentage of germination. In supporting to our result, scientist reported that 20% of seaweed extract shown maximum percentage of seed germination^{17, 20}. Control shown maximum of 60% of germination, the control medium was supplemented with modified MS salts, 30% sucrose and without seaweed extract (Fig. 1).

Fig. 1: Effect of Sargassum polycystum on in vitro germination of Cowpea seeds

Multiple shoot initiation:

Cotyledonary node explants were collected from 3 days old seedlings, the excised explants were cultured on to the medium supplemented with various concentrations of TDZ (0.5-2.5 mg/L) and Kinetin (0.2-1.0 mg/L). Medium supplemented with TDZ 1.5 mg/L and Kin 0.6 mg/L shown maximum percentage (90%) of response with mean 10.2 number of shoots (Table 1). As the concentration of TDZ increases, percentage of response decreases. Shoot organogenesis of some crops in tissue culture has been achieved recently using thidiazuron, a substituted phenyl urea compound with cytokinin activity. The highest number of shoots (4.44) was produced by explants with both entire cotyledons at 1 mg/L of kinetin³. According to Margara (1969)²¹, BAP and Kinetin stimulate formation and development of buds. In contrast in V. radiata the effect of NAA in shoots multiplication was statistically equivalent to cytokinins especially BAP²².

To increase the multiple shoot induction, plant growth regulators were tested in combination with the seaweed. The best responded concentration of TDZ and Kin (1.5 mg/L and Kin 0.6 mg/L) were tested along with various concentrations (5-25%) of Sargassum polycystum. Of the concentration tested, medium fortified with 1.5 mg/L TDZ, 0.6 mg/L Kin and 20% of seaweed extract shown best response and produced mean 14.8 shoots per explant. Increase in the response is due to the presence of more than one type of plant growth regulators (Table 2). Crouch and van staden (1991)²³ reported the presence of IAA from the auxin-type active fractions in the extract obtained from Ecklonia maxima. Within these active fractions, indole-3- carboxylic acid, indole-3-aldehyde, and other indole compounds were also reported²⁴.

The medium was composed of MS salts and B5 vitamins, 3% sucrose, 05-2.5 mg/L of TDZ and 0.2-1.0 mg/L of Kin. Each experiment was repeated thrice. Data were recorded after 3 subculture. Means followed by the same letter within columns are not significantly different, according to Duncan’s multiple range test (P<0.05). Best results are indicated in bold. (Table-2).

Table 1: Effect of TDZ and Kinetin on multiple shoot initiation from cotyledonary node explant

Concentration of plant growth regulators (mg/L)		Percentage of response (%)	Mean number of shoots	Mean shoot length (cm)
TDZ	KIN	Percentage of response (%)	Mean number of shoots	Mean shoot length (cm)
0.5	0.2	65	7.2±0.15^d	2.8±0.60^d
1.0	0.4	80	8.6±0.23^b	4.2±0.43^b
1.5	0.6	90	10.2±0.46^a	4.9±0.18^a
2.0	0.8	70	7.9±0.35^c	3.4±0.16^c
2.5	1.0	45	4.8±0.18^e	2.1±0.13^e

Table 2: Influence of PGR’s and Sargassum polycystum on multiple shoot initiation

Concentration of plant growth regulators (mg/L)	*Concentration of Sargassum polycystum* extract (%)**	Percentage of response (%)	Mean number of shoots	Mean shoot length (cm)
TDZ + KIN		Percentage of response (%)	Mean number of shoots	Mean shoot length (cm)
1.5+0.6	5	85	12.3±0.13^b	6.1±0.53^b
	10	100	14.8±0.21^a	6.7±0.11^a
	15	75	10.1±0.35^c	5.3±0.18^c
	20	55	8.5±0.16^d	3.9±0.12^d
	25	30	4.2±0.28^e	3.2±0.29^e

The medium was composed of MS salts and B5 vitamins, TDZ 1.5 mg/L, Kin 0.6 mg/L, 3% sucrose and seaweeds at different concentrations. Each experiment was repeated thrice. Data were recorded after 3 subculture. Means followed by the same letter within columns are not significantly different, according to Duncan’s multiple range test (P<0.05). Best results are indicated in bold

Shoot elongation using seaweed extract:

Shoots of about 5-6cm in length was excised and inoculated in to the medium containing MS salts and various concentration (5-25%) of S. Polycystum. In support to our result, Craigie in 2011²⁵ reported that the plant hormones from brown seaweeds, such as cytokinins, auxins and gibberellins, have been shown to enhance germination, growth and yield in crop plants²⁶. So we believe that the enhanced activity of Sargassum polycystum may be due to the presence of plant hormones. Medium supplemented with 15% of seaweed extract exhibited maximum of 12.6 com of shoot length with 100% response (Table 3).

Table 3: Effect of Sargassum polycystum on shoot elongation

*Concentration of S. polycystum* extract (%)**	Percentage of response (%)	Mean shoot length (cm)
5	70	8.8±0.26^d
10	90	10.4±0.12^b
15	100	12.6±0.33^a
20	80	9.3±0.15^c
25	60	6.2±0.12^e

The medium was composed of MS salts and B5 vitamins, 3% sucrose and seaweeds at different concentrations. Each experiment was repeated thrice. Data were recorded after 2 subculture. Means followed by the same letter within columns are not significantly different, according to Duncan’s multiple range test (P<0.05). Best results are indicated in bold

Rooting and Hardening:

After a subculture, medium exposed portion of the elongated shoots were excised and inoculated in the medium supplemented with 0-25% of seaweed extract. The control medium (without seaweed extract) shown least percentage of response and produced 7.7 mean number of roots (Fig. 2). Our results are correlating with the results of Machuka et al. (2000)²⁷, they reported that regenerated cowpea shoots showing poor root formation on hormone-free medium. Crouch and van Staden (1991)²³ also showed a stimulation of rooting in the presence of a concentrated extract prepared from brown algae. The plantlets were transferred to plastic cups, as the leaves are young and soft, it became easily dried when exposed to temperature above 27°C. So, the young plants which were kept in the controlled conditions for 3 days shown higher survival rate of 85%.

Fig. 2: Effect of Sargassum polycystum on rooting of elongated shoots

The medium was composed of MS salts and B5 vitamins, 3% sucrose and seaweeds at different concentrations. Each experiment was repeated thrice. Data were recorded after a subculture

ACKNOWLEDGEMENT:

The authors are grateful to the Department of Plant Science, Bharathidasan University for their support in algal collection.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Nielson SS, Brandt WE, Singh BB. Genetic variability for nutritional composition and cooking time of improved cowpea lines. Crop Sci. 1993; 33: 469-472.

2. Sani LA, Usman IS, Ishiyaku MF, SM Bugaje. Optimization of protocol for multiple shoots regeneration apical meristem of embryonic axes in cowpea (Vigna unguiculata L. Walp), Bayero Journal of Pure and Applied Sciences. 2017; 10(1): 428 – 433

3. Diallo MS, Ndiaye A, Sagna M, Gassama-Dia YK. Plants regeneration from African cowpea variety (Vigna unguiculata L. Walp.). African Journal of Biotechnology. 2008; 7 (16):2828-2833.

4. Sahoo L, Sugla T, Singh ND, Sonia, Nijsure P,Gulati A, Singh RP, Jaiwal PK. Current status and future strategies in genetic improvement of cowpea. Vegetal Res. 2001; 28: 9–16.

5. Kartha KK, Pahl K, Leung NL, Mroginski LA. Plant regeneration from meristems of grain legumes: soybean, cowpea, peanut, chickpea, and bean. Can. J. Bot. 1981; 59: 1671–1679.

6. Kulothungan S, Ganapathi A, Shajahan A, Kathiravan K. Somatic embryogenesis in cell suspension culture of cowpea (Vigna unguiculata (L.) Walp. Isr. J. Plant Sci. 1995; 43: 385–390.

7. Nagl W, Ignacimuthu S, Becker J. Genetic engineering and regeneration of Phaseolus and Vigna. State of the art and newattempts. J. Plant Physiol. 1997; 150: 625–644.

8. Brar MS, Al-Khayri JM, Morelock TE, Anderson EJ. Genotypic response of cowpea Vigna unguiculata (L.) to in vitro regeneration from cotyledon explants. In Vitro Cell. Dev. Biol. 1999; 35: 8–12.

9. Le BV, Cruz de Carvalho MH, Zuily-Fodil Y, Thi ATP, Van KTT. Direct whole plant regeneration of cowpea (Vigna unguiculata (L.)Walp) from cotyledonary node thin cell layer explants. J. Plant Physiol. 2002; 159: 1255–1258.

10. Mao JQ, Zaidi MA, Arnason JT, Altosaar I. In vitro regeneration of Vigna unguiculata (L.) Walp. cv. blackeye cowpea via shoot organogenesis. Plant Cell, Tissue Organ Cult. 2006; 87: 121–125.

11. Prem Anand R, Ganapathi A, Ramesh A, Vengadesan G, Selvaraj N. High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of cowpea (Vigna unguiculata L. Walp). In Vitro Cell. Dev. Biol. — Plant. 2000; 36: 475–480.

12. Baskaran P, van Staden J. Somatic embryogenesis of Merwilla plumbea (Lindl.) Speta. Plant Cell Tissue Organ Cult. 2012; 109:517–524

13. Khan W, Rayirath UP, Subramanian S, Jithesh MN, Rayorath P, Hodges DM, CritchleyAT, Craigie JS, Norrie J, Prithivraj B. Seaweed extracts as biostimulants of plant growth and development. Plant Growth Regul. 2009; 28:386–399.

14. Raveendar S, Premkumar A, Sasikumar S, Ignacimuthu S, Agastian P. Development of a rapid, highly efficient system of organogenesis in cowpea Vigna unguiculata (L.) Walp South African Journal of Botany. 2009; 75: 17–21

15. Durand N, Briand X, Meyer C. The effect of marine bioactive substances (NPRO) and exogenous cytokinins on nitrate reductase activity in Arabidopsis thaliana. Physiol Plant 2003; 119: 489–493

16. Stirk WA, Arthur GD, Lourens AF, Novak O, Strnad M, van Staden J. Changes in cytokinin and auxin concentrations in seaweed concentrates when stored at an elevated temperature. J Appl Phycol 2004; 16:31–39.

17. Kumar G, Sahoo D. Effect of seaweed liquid extract on growth and yield of Triticum aestivum var. Pusa Gold. J Appl Phycol. 2011; 23:251–255

18. Crouch IJ, van Staden J. Evidence for the presence of plant growth regulators in commercial seaweed products. Plant Growth Regul. 1993; 13: 21–29

19. Tarakhovskaya ER, Maslov Yu I, Shishova MF. Phytohormones in algae. Russ J Plant Physl. 2007; 54:163–170.

20. Vinoth S, Gurusaravanan P, Jayabalan N.Effect of seaweed extracts and plant growth regulators on high-frequency in vitro mass propagation of Lycopersicon esculentum L (tomato) through double cotyledonary nodal explants. J Appl Phycol. 2012; 24:1329–1337.

21. Margara J. Etudes des facteurs de la néoformation de bourgeons in vitro chez le chou-fleur, Brassica oleracea L. Var botrytis. Ann. Physiol. Vég, INRA. 1969; 11: 95-112.

22. Gulati A, Jaiwal PK. Plant regeneration from cotyledonary node explants of mungbean (Vigna radiata L. Wilczek). Plant cell Rep 1994; 13: 523-527.

23. Crouch U, Van Staden J. Evidence for rooting factors in a seaweed concentrate prepared from Ecklonia maxima. J Plant Physiol. 1991; 137:319–322.

24. Briceño-Domínguez D, Hernández-Carmona G, Moyo M, Stirk W, van Staden J. Plant growth promoting activity of seaweed liquid extracts produced from Macrocystis pyrifera under different pH nd temperature conditions. J Appl Phycol. 2014; DOI 10.1007/s10811-014-0237-2

25. Craigie JS. Seaweed extract stimuli in plant science and agriculture. J Appl Phycol 2011; 23:371–393

26. Ali N, Farrell A, Ramsubhag A, Jayaraman J. The effect of Ascophyllum nodosum extract on the growth, yield and fruit quality of tomato grown under tropical conditions. J Appl Phycol. 2015; DOI 10.1007/s10811-015-0608-3.

27. Machuka J, Adesoye A, Obembe OO. Regeneration and genetic transformation in cowpea. Proceedings of World Cowpea Conference III. IITA, Ibadan, Nigeria, 2000 pp. 185–196.

Received on 02.08.2018 Modified on 22.10.2018

Research J. Pharm. and Tech. 2019; 12(4):1580-1584.

DOI: 10.5958/0974-360X.2019.00262.2