Optimization of micropropagation protocol for three cotton varieties regenerated from apical shoot

Biochemistry Unit, Department Chemical Sciences, College of Natural and Applied Science, Fountain University Osogbo, Nigeria. Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria. Department of Biochemistry, Igbinedion University Okada, Nigeria. Faculty of Pharmaceutical Sciences, University of Ilorin, Ilorin, Nigeria. Agricultural Research Council, Vegetable and Ornamental Plant Institute, Pretoria, South-Africa.


INTRODUCTION
The focus of research in plant cell culture for many crop species was to be able to put species into tissue culture maintain or grow the plant cells, tissues or organs under sterile controlled laboratory conditions and ultimately regenerate a normal fertile plant.In comparison with other crops, successes in cotton tissue culture lag behind those of other crops.In vitro cultured cotton cells have been induced to undergo somatic embryogenesis in numerous laboratories using varied strategies (Shoemaker et al., 1986;Chen et al., 1987;Trolinder and Goodin, 1987;Kolganova et al., 1992;Zhang, 1994a;Zhang et al., 1996Zhang et al., , 1999)).
Regenerated plants have been obtained from explants such as hypocotyls, cotyledon, root (Zhang, 1994a) and from various cotton species (Zhang, 1994b).Gould et al. (1991) reported a successful regeneration method of two cotton varieties; G. barbadense cultivars and G. hirsutum cultivars that was independent of genotype; however, rooting efficiency was low.Nasir et al. (1997), Morre et al. (1998) and Zapata et al. (1999) also reported the regeneration of cotton plants from shoot meristems.This method has also been successfully used in cotton transformation when combined with particle bombardment (McCabe and Martinell, 1993).Trolinder and Goodin (1987) reported regeneration of cotton plants from callus by somatic embryogenesis, and the efficiency of regeneration via somatic embryogenesis has been reported to improve significantly in recent years (Trolinder et al., 1989;Rajasekaran et al., 1996;Zhang et al., 2001), some difficulties still remain.Major limitations had been that only few cultivars can be induced to produce somatic embryos and regenerative plants.Most responsive lines are Coker varieties, which are no longer under cultivation (Feng et al., 1998).Aside from the genotype limitation, many of the plants regenerated from callus as somatic embryos are abnormal (Cousins et al., 1991;Trolinder and Goodin, 1987;Rajasekaran et al., 1996).Due to these shortcomings, cotton biotechnology has been a major task in cotton breeding and production.As an improved approach, Renfroe and Smith (1986) reported regeneration of cotton from isolated shoot meristem from seedlings of G. hirsutum L. cv. to obtain regenerated plants.Gould et al. (1991) extended this approach by using two G. barbadense cultivars and 19 G. hirsutum cultivars in his research, which showed that regeneration from shoot tips was genotype-independent. Saeed et al. (1997), Morre et al. (1998) and Zapata et al. (1999) also reported the regeneration of cotton plants from shoot meristems.However, rooting efficiencies were low in these reports (from 38 to 58%).In this report, an optimized regeneration protocol with improved rooting efficiency in shoot apex based cotton regeneration system is presented.Three factors that could affect the rooting efficiency of shoot apices were investigated in this research: 1) Effect of seed sterilization method, 2) Effect of shoot apex age, and 3) Effect of concentration of IAA shock.In the end, an improved regeneration protocol with rooting efficiency up to 87% was developed.The protocol uses cotton shoot apices as explants and combines basic rooting, IAA shock and grafting steps to increase rooting efficiency up to 47% and regeneration to 87%.

Seed disinfection methods
Cotton seeds were de-linted in concentrated sulphuric acid (H 2 SO 4 ) then washed in tap water.The de-linted seed were then wrapped in cheese cloth and soak in tap water for 1 h.Cotton seeds were disinfected via four methods: Method 1: Cotton seeds were treated with 70% ethanol for 2 min prior to a 20 min exposure to 10% Clorox ® (5.25% sodium hypochlorite (NaOcCl)) solution with two drops of Tween 20 per 100 ml, and rinsed three times with sterile double-distilled water.The seeds were then placed on seed germination medium.Method 2: Cotton seeds were treated with a 50% Clorox ® (5.25% NaOcCl) solution with two drops of Tween 20 per 100 ml on a rotary shaker at 50 rpm for 20 min and rinsed at least three times with sterile double-distilled water.The seeds were then placed on seed germination medium.Method 3: Cotton seeds were treated with 20% hydrogen peroxide for 2 h and rinsed three times with double-distilled water.The seeds were then placed overnight on a rotor shaker at 100 rpm.After removing the seed coat, the seeds were then placed on seed germination medium.Method 4: Cotton seeds were treated as described in Afolabi-Balogun et al. (2011).After removing the seed coat, the seeds were placed on seed germination medium.

Seed germination
Three seeds were placed in each germination media (Afolabi-Balogun et al., 2011) and incubated in the dark at 28°C overnight and then in the light for 5 days.Upon removal from incubation, the number of elongated shoots was counted.Contamination was determined by visual inspection for fungal and/or bacterial growth.

Shoot apex isolation
The seedling apexes were isolated as described by Afolabi-Balogun et al. (2011).The epicotyl (shoot) was placed on MS+Kin medium.The plants were kept in growth room at 27± 2°C to 16 h light and 8 h dark at 70% humidity.The plantlets were grown for 10 days.

Shoot elongation and rooting development
Thirty shoots from each variety without root development were subjected to IAA shock at concentrations 0.1, 0.5, 1.0, 1.5 and 2.0 mg/ml for one minute.The treated shoots were rinsed and transferred to fresh MS medium for another three weeks.The number of rooted plants was recorded and the rooted plants were transferred to Magenta boxes containing MS medium and incubated in a culture chamber for four weeks before being transferred to the greenhouse.

Plantlets graft
Grating of un-rooted elongated shoots from MS medium after IAA shock onto the seedling stocks of the same variety was done by cutting the bottom of the scion into a wedge with a scalpel blade, then the upper part of the seedling stocks was cut under the first true leaf; and a slit (about 1.0 cm) on the stem was cut vertically.The decapitated end of the root stocks and matching cut ends of the scions were treated with 0.2 mg/L IAA + 0.1 mg/L GA. for 5 min.Then the treated scion was inserted into the slit and the cambiums were lined up.The grafted plant was then covered by a 1000 ml flask and kept in a humid chamber for a week.After which the flask was removed and the plants kept in the humid chamber for another week before being transferred to the greenhouse.

Data collection and analysis
Data were obtained at 25-30 days and at 42 days on the number viable shoots and viable shoots with roots.Other observations made were on shoot health (on a rating scale 1 to 5 scale, with 1 being poorest chlorophyll development, and 5, best chlorophyll development), leaf abscission (on a 1 to 5 rating scale with a rating of 5 being complete leaf retention, and 1 complete leaf abscission), number of explants with callus, relative calli size, root length, and branching.Data was analyzed as a completely randomized design with three replications using ProcGLM of SAS program (SAS, 1987).Means of statistically significant (p=0.05)treatments were separated using LSD.

Seed disinfection methods
The extent of sterility was measured be physical examination of the culture bottle for contaminant such as mould.Maximum surface sterilization was observed with the seed disinfected with method 4 (number of contaminated seed is zero) (Figure 1).Methods 1 and 2 did not give perfect sterilization.Use of only 50% Clorox ® gives the least sterilization.Combining Clorox ® and hydrogen peroxide gave a better result, but this was still not as efficient as hydrogen peroxide.

Seed germination
All cotton seeds varieties germinated on MS though seedlings elongation on germination medium was very slow.For enhancement of growth, the tiny seedlings were transferred to different media supplemented with plant growth regulators (BAP, NAA, IBA) and activated charcoal (AC).The highest rate of elongation was achieved on MS supplemented with 2.5 mg/L BAP + 0.1% (w/v) AC.

Shoot apex isolation
Vigorous shoot growth was observed when kinetin is supplemented to the media.

Shoot elongation and rooting development
The rooting efficiency of the three varieties was significantly different in different concentrations of IAA (p=0.027)(Figure 2).The effect of different IAA shock concentrations varied from 6.7 to 47%.The highest efficiency (47%) was observed for a 1.5 mg/ml IAA and the lowest efficiency (12%) was observed for 0.1 mg/ml IAA.So the concentration of 1.5 mg/ml IAA was chosen for regeneration.The rate of rooting of elongated shoots cultured on various media is presented in Table 1.Optimum rooting was observed using ERM 4 which was about (47%) while the lowest rooting was observed with ERM 2 giving only (6.7%).Hence, a concentration of 1.5 mg/ml IAA was chosen in the regeneration system.The difference of rooting efficiency was not significantly different in all varieties (p=0.08).This result indicated that rooting efficiency is genotype independent.

Plantlet grafting
Rooting efficiency of plantlet as well as survival rate was improved to eighty five percent when plantlets were kept humid, pre-treating the scion and stock with 0.1 nmg/L IAA + 0.2 mg/L GA (Figure 3).Grafting is a very useful technique and is commonly used in horticultural crops.

DISCUSSION
Recently, several researchers have regenerated plants from shoot tip meristems (Zapata et al., 1999).Gould et al. (1991) reported that the yield of shoots in vitro from isolated apices depends on the incidence of contamination and rooting efficiency.In recent years, protocols involving proliferation of cotton shoots (Agrawal et al., 1997;Hemphill et al., 1998) have been published.The rooting efficiency ranged from 38 to 58% in their reports.Here we report an optimized regeneration protocol involving shoot tips regenerated directly without a callus phase, this method has the advantage of being genotype-independent; almost all cultivars can be regenerated from shoot tips.The use of shoot tips as explants in an Agrobacterium-mediated transformation system is a good way to overcome the obstacles in traditional Agrobacterium-mediated transformation.From the germination results, all seeds sterilized by hydrogen peroxide germinated in 5 days (Figure 1); seeds sterilized by both Clorox ® methods had a lower germination rate (95 and 37%, respectively).The reason for those results may be that the residual of Clorox, specifically, chlorine, suppressed the germination of cotton seeds, while the residual of hydrogen peroxide is water and CO 2 , did not affect the germination of cotton seeds.
The age of explants has a significant effect on shoot tip elongation (Table 2).The elongation rates of the three varieties were not significantly different from each other (p=0.1573).The elongation rate was also affected by the size of isolated tips.It was observed that if the starting size of the apex was less than 1 mm, the tips would not grow at all.
The efficiency of the rooting media was evaluated based on the increase in length and number of roots developed per seedling.The highest rate of elongation was achieved on MS supplemented with 2.5 mg/L BAP + 0.1% (w/v) AC however, MS + 3% (w/v) sucrose + 1.5 mg/IAA proved more effective for the development of better root system and the rooting of the plantlet was by grafting procedure.
The type and concentration of plant growth regulators     strongly influenced the organogenic potential of the apical shoot explant.The responding frequency of shoot seemed to depend more on concentration of indole-3acetic acid (IAA).The regeneration response of apical shoot explant and split cotyledon node from cotton to the concentration of IAA is shown in Table 3.It is evident that without IAA regeneration of apical shoot is low and maximum shoot regeneration response has been observed with 1.5 mg/L IAA concentration along with MS.
The further increment in IAA concentration to 2.0 mg/L along with MS showed decreased shoot regeneration response.We observed maximum number of shoots when GA was combined with IAA in all variety.

Figure 1 .
Figure 1.Effect of sterilization method on seed germination.

Figure 2 .Figure 3
Figure 2. Effect of IAA shock on stimulating the rooting of previously unrooted Cotton shoot apices.Vertical bar represents the standard error of the 5 treatments of IAA.

Table 1 .
Elongation and rooting media composition used in optimization.

Table 2 .
Mean number of explants elongated on elongation medium from 3 cotton varieties at 4 different ages.
a + Different letter label significant at p=0.05 level using LSD method, ++ Mean±Std.

Table 3 .
Regeneration response of apical shoot explant and split cotyledon node from cotton to the concentration of IAA.Means followed by the same letter are not significantly different at p=0.05.Glu-Glucose, Suc-Sucrose, Ac.Char-Activated Charcoal.SA-Shoot apices SCN-Splited Cotyledon Node. a