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AN EFFICIENT AND REPRODUCIBLE TISSUE CULTURE PROCEDURE FOR CALLUS INDUCTION AND MULTIPLE SHOOTS REGENERATION IN GROUNDNUT (Arachis hypogaea L.).

Byline: N. Ahmad, M. R. Khan, S. H. Shah, M. A. Zia, I. Hussain, A. Muhammad and G. M. Ali

Keywords: Callus induction, Embryo slices, Genetic transformation, Multiple shoots, Plant growth regulators

INTRODUCTION

Groundnut (Arachis hypogaea L.) is the most dominant oilseed crop and is ranked 3rd among oil producing crops in the world (Upadhyaya et al., 2003). Groundnut is mainly cultivated throughout the world with specific regions like tropical, sub-tropical, and temperate areas (Cuc et al., 2008). Plant breeding of groundnut is very difficult, time consuming and requires highly skilled labors to perform emasculation, crossing and selection due to their self-pollination and narrow genetic base behaviors (Pasupuleti et al., 2013; Moretzsohn et al., 2005). Gene transformation limits all these hurdles and introduces important genes of good agronomic characteristics (Tiwari et al., 2008; Lemaux, 2008). But an efficient regeneration method is prerequisite for genetic transformation approach. So, the establishment of an adaptive tissue culture system for groundnut would be very useful for improved production with good seed quality (Sharma and Anjaiah, 2000).

Successful plants regeneration system relies on many factors such as plants growth regulators, composition of media, genotype, explants, photoperiod, temperature and the environment (Ishag et al., 2009; (Shah et al., 2013; Shah et al., 2014 a-b). BAP along with NAA promoted callus induction; while TDZ and BAP were reported for initiation of multiple shoot cultured explants (Hutchinson et al., 1994). In the past groundnut in vitro regeneration was conducted by using the whole immature cotyledon (Robinson et al., 2011, Palanivel et al., 2002), cotyledonry nodes and mature embryos (Lacroix et al., 2003), mature dry seeds (Baker et al., 1995), leaflets (Chengalrayan et al., 2001; Tiwari and Tuli, 2009), hypocotyls (Venkatachalam et al., 1997), mature epicotyl (Shan et al., 2009). However, it is difficult to obtain more planting material in short time due to the larger size of these explants.

In the past decades, tissue culture technique has long been applied to achieve somaclonal variation by plant breeders (Kaeppler et al., 2000). Selection of plant species through somoclonal variations that are used to create genetic variability among crop plants and the struggles are being done to improve crop yield, oil contents, and cultivars development of Arachis hypogaea that have resistance against various diseases (Robinson et al., 2011). The plant breeders have been struggling for a long time to improve groundnut yield, quality and to develop resistant varieties for pest, drought, fungus, cold and salt but unfortunately, limited success has been achieved by conventional breeding (Banerjee et al., 2007). While genetic transformation has become a popular tool for transferring desirable genes into crops without any barrier in less time as compared to conventional plant breeding (Taji et al., 2002).

But an efficient tissue culture protocol is pre-requisite for successful genetic transformation. Therefore, we planned this study to optimize an efficient and reproducible tissue culture procedure for callus induction, multiple shoot regeneration and in vitro root induction in groundnut.

MATERIALS AND METHODS

Plant material, disinfection and explants preparation: The seeds of two groundnut cultivars namely Golden and BARI 2001 were taken from Barani Agricultural Research Institute (BARI), Chakwal, Pakistan. Seventy percent ethanol was used for surface sterilization of mature and healthy seeds. Surface sterilization was done for two minutes with 70% ethanol and then for fifteen minutes with 6% sodium hypochlorite (NaOCl) along with continuous shaking. Then their seeds were washed with double distilled water until the removal of traces of ethanol and NaOCl. These seeds were put in double distilled water for one and half hour to facilitate zygotic embryo excision. Embryos slice; as a source of explants was removed aseptically from their seeds by ripping out the seed coat. Through bilateral cutting of these seeds, very thoroughly radicle and plumule were excised and then their embryos were cut into several pieces.

Plant growth regulators and culture conditions: This research study was conducted at NIGAB, NARC, Islamabad. Murashige and Skoog (1962) basal media with vitamin at pH 5.8 fortified with 30 g/l sucrose and 3 g/l gum powder was used in all the cultures. Various levels of cytokinin (BAP, KIN) and auxins (NAA, TDZ, IAA) on MS medium were used to obtain callus and multiple shoots regeneration (Tables 1 and 2). In vitro root induction was achieved with the application of post autoclaved IBA in MS media.

Effect of growth regulators on callus and multiple shoot induction: MS basal media having various levels of BAP, IAA and NAA was used for callus induction (Table 1). Then explants were placed in contact with the media under tissue paper to protect them from high light intensity at 28 AdegC. The calli were induced and explants were expanded five times in their original size after ten days. Subsequently, the explants were sub cultured for more ten days on the same media for further regeneration. The regenerated calli continued to proliferate and then sub-culturing on multiple shoot induction media (MSM), having hormonal combinations of BAP, KIN and TDZ (Table 2) for 10-15 days under sixteen hours' photoperiod having fluorescence light intensity of 50 umolm-2s-1 and 65-70% relative humidity at 25 +- 2 AdegC (Shah et al., 2015; Shah et al., 2020).

In vitro rooting and plantlet acclimatization: Individual shoots (2-5 cm in length) were aseptically cut from multiple shoots and were put on growth regulator free MS fortified by IBA for 2-3 weeks and repeated three times (Table 3). Data was recorded after four weeks for rooted plants, No. of roots/explant and root length. Then these were transferred to hydroponics condition in the glass house containing Yoshida solution (Yoshida et al., 1976) for further development of roots for 10 days. For acclimatization maintenance of plantlets in a combination of manure and sand (1:3) at a constant temperature of 30 AdegC in a glass house for fourteen days after profuse root elongation (about 3 cm) with secondary roots.

Statistical analysis: The whole experimental trials were arranged using completely randomized design. The significant difference was noticed by using ANOVA technique at Pa$?0.05 and Duncan's multiple range test was applied to check the significant differences among means. For this purpose, statistics software namely The Statistix v. 8.1 was used (Analytical Software, 2005).

Table 1. Callus induction media used for callus proliferation in groundnut.

Media###Composition

CIM1###MS + BAP (1.0 mg/l)

CIM2###MS + BAP 2.0 mg/l

CIM3###MS + BAP 3.0 mg/l

CIM4###MS + BAP 4.0 mg/l

CIM5###MS + BAP 5.0 mg/l

CIM6###MS + IAA (2.0 mg/l) + BAP (3.5 mg/l)

CIM7###MS + IAA (2.5 mg/l) + BAP (4.5 mg/l)

CIM8###MS + IAA (3.0 mg/l) + BAP (5.5 mg/l)

CIM9###MS + IAA (3.5 mg/l) + BAP (6.5 mg/l)

CIM10###MS + IAA (4.0 mg/l) + BAP (7.5 mg/l)

CIM11###MS + NAA (0.5 mg/l) + BAP (3.5 mg/l)

CIM12###MS + NAA (1.0 mg/l) + BAP (4.5 mg/l)

CIM13###MS + NAA (1.5 mg/l) + BAP (5.5 mg/l)

CIM14###MS + NAA (2.0 mg/l) + BAP (6.5 mg/l)

CIM15###MS + NAA (2.5 mg/l) + BAP (7.5 mg/l)

Table 2. Multiple shoot induction media used for multiple shoot formation in groundnut.

Media###Composition

MSM1###MS + BAP (4.0 mg/l)

MSM2###MS + BAP (4.5 mg/l)

MSM3###MS + BAP (5.0 mg/l)

MSM4###MS + BAP (5.5 mg/l)

MSM5###MS + BAP (6.0 mg/l)

MSM6###MS + BAP (4.0 mg/l) + Kin (0.5 mg/l)

MSM7###MS + BAP (4.5 mg/l) + Kin (1.0 mg/l)

MSM8###MS + BAP (5.0 mg/l) + Kin (1.5 mg/l)

MSM9###MS + BAP (5.5 mg/l) + Kin (2.0 mg/l)

MSM10###MS + BAP (6.0 mg/l) + Kin (2.5 mg/l)

MSM11###MS + BAP (4.0 mg/l) + TDZ (0.5 mg/l)

MSM12###MS + BAP (4.5 mg/l) + TDZ (1.0 mg/l)

MSM13###MS + BAP (5.0 mg/l) + TDZ (1.5 mg/l)

MSM14###MS + BAP (5.5 mg/l) + TDZ (2.0 mg/l)

MSM15###MS + BAP (6.0 mg/l) + TDZ (2.5 mg/l)

Table 3. Root induction media used for root formation in groundnut.

Media###Composition

RIM1###MS + IBA (0.5 mg/l) (IBA used after autoclaving)

RIM2###MS + IBA (1.0 mg/l) (IBA used after autoclaving)

RIM3###MS + IBA (1.5 mg/l) (IBA used after autoclaving)

RIM4###MS + IBA (2.0 mg/l) (IBA used after autoclaving)

RIM5###MS + IBA (2.5 mg/l) (IBA used after autoclaving)

RIM6###MS + IBA (0.5 mg/l) (IBA used after filter sterilization)

RIM7###MS + IBA (1.0 mg/l) (IBA used after filter sterilization)

RIM8###MS + IBA (1.5 mg/l) (IBA used after filter sterilization)

RIM9###MS + IBA (2.0 mg/l) (IBA used after filter sterilization)

RIM10###MS + IBA (2.5 mg/l) (IBA used after filter sterilization)

Table 4. Assessment of various combinations of PGRs on callus induction in groundnut.

Callus induction###GOLDEN###BARI 2001

media###No. of explants###Callus induction (%)###No. of explants###Callus induction

###responded (%)###responded (%)###(%)

CIM1###13.3 +- 1.5###6.3 +- 1.5###10.6 +- 2.5###5.3 +- 1.5

CIM2###23.5 +- 3.0###10.3 +- 1.7###17.3 +- 2.8###8.0 +- 2.2

CIM3###35.6 +- 3.7###13.5 +- 2.3###26.2 +- 3.9###10.3 +- 2.1

CIM4###45.3 +- 3.9###19.7 +- 3.4###33.3 +- 3.9###13.2 +- 2.3

CIM5###38.1 +- 4.1###15.5 +- 3.1###26.6 +- 3.1###9.0 +- 1.8

CIM6###45.6 +- 4.3###40.6 +- 3.8###40.5 +- 4.0###33.3 +- 3.2

CIM7###57.3 +- 5.3###51.3 +- 4.0###49.9 +- 4.4###38.5 +- 4.1

CIM8###68.1 +- 5.6###58.1 +- 4.2###53.6 +- 4.3###41.7 +- 4.4

CIM9###61.0 +- 5.2###43.7 +- 3.9###56.4 +- 5.2###39.3 +- 4.5

CIM10###50.4 +- 4.8###39.5 +- 4.3###45.2 +- 4.2###30.2 +- 4.3

CIM11###58.8 +- 4.9###45.3 +- 4.7###46.7 +- 4.7###38.8 +- 3.2

CIM12###74.7 +- 5.3###66.3 +- 4.3###70.7 +- 5.1###61.3 +- 4.4

CIM13###92.2 +- 5.5###86.6 +- 5.5###88.3 +- 5.3###78.3 +- 4.8

CIM14###86.2 +- 5.1###70.4 +- 4.9###75.4 +- 5.0###60.7 +- 4.3

CIM15###73.7 +- 4.8###61.3 +- 4.7###61.7 +- 4.4###49.5 +- 4.9

Table 5. Assessment of various combinations of PGRs on multiple shoot formation in groundnut.

Multiple shoot###GOLDEN###BARI 2001

induction media###No. of explants###No. of shoots/ explants###No. of explants###No. of shoots/

###responded (%)###responded (%)###explants

MSM1###44.7 +- 3.7###1.4 +- 0.5###40.4 +- 3.3###1.3 +- 0.5

MSM2###50.5 +- 3.9###1.9 +- 0.6###48.6 +- 3.7###1.9 +- 0.7

MSM3###71.3 +- 5.5###2.7 +- 0.7###64.6 +- 5.3###2.5 +- 0.9

MSM4###65.3 +- 5.3###3.4 +- 0.4###60.3 +- 3.1###3.1 +- 0.8

MSM5###59.6 +- 4.2###3.1 +- 0.3###49.7 +- 4.2###1.9 +- 0.4

MSM6###51.4 +- 3.1###2.1 +- 0.7###45.6 +- 4.1###1.5 +- 0.6

MSM7###63.7 +- 4.4###3.3 +- 0.8###56.3 +- 4.3###3.0 +- 0.8

MSM8###65.2 +- 5.2###4.6 +- 0.6###69.5 +- 4.3###4.1 +- 0.6

MSM9###80.1 +- 5.8###4.1 +- 0.3###62.7 +- 4.7###3.6 +- 0.5

MSM10###71.6 +- 5.8###3.7 +- 0.3###59.4 +- 4.0###2.4 +- 0.4

MSM11###59.9 +- 4.2###3.2 +- 0.6###53.3 +- 4.3###3.3 +- 0.8

MSM12###70.5 +- 5.5###5.0 +- 0.7###58.8 +- 5.0###3.7 +- 0.9

MSM13###88.3 +- 5.1###9.2 +- 1.1###74.2 +- 5.3###7.1 +- 1.2

MSM14###83.7 +- 5.9###6.3 +- 1.1###68.3 +- 5.6###5.2 +- 1.1

MSM15###71.5 +- 5.5###3.7 +- 0.5###56.5 +- 5.1###3.2 +- 0.9

Table 6. Assessment of various combinations of PGRs on number of leaves per explant and average height of shoots (cm) in groundnut.

Multiple shoot###GOLDEN###BARI 2001

induction media###No. of leaves per###Average height of shoots###No. of leaves per###Average height of

###explants###(cm)###explants###shoots (cm)

MSM1###3.3 +- 0.8###2.1 +- 0.8###3.1 +- 0.7###2.2 +- 0.4

MSM2###3.5 +- 0.8###2.5 +- 0.9###3.6 +- 0.5###2.2 +- 0.4

MSM3###3.9 +- 0.3###3.8 +- 0.6###3.8 +- 0.8###3.4 +- 0.9

MSM4###4.2 +- 0.3###3.6 +- 0.7###4.0 +- 1.1###3.1 +- 0.8

MSM5###3.3 +- 0.9###3.1 +- 0.7###3.1 +- 0.6###2.9 +- 0.8

MSM6###3.4 +- 0.3###2.5 +- 0.8###3.3 +- 0.9###2.1 +- 0.5

MSM7###3.8 +- 0.8###3.0 +- 0.7###3.9 +- 0.9###2.8 +- 0.8

MSM8###4.4 +- 0.8###4.8 +- 0.6###4.1 +- 0.7###4.3 +- 0.6

MSM9###4.2 +- 0.7###4.3 +- 0.6###3.8 +- 0.8###3.9 +- 1.1

MSM10###3.3 +- 0.3###3.0 +- 0.9###3.3 +- 0.9###3.5 +- 0.9

MSM11###3.0 +- 0.4###2.8 +- 0.4###3.1 +- 0.7###2.8 +- 0.4

MSM12###3.3 +- 0.3###3.1 +- 0.3###3.4 +- 0.5###2.8 +- 0.6

MSM13###6.1 +- 1.3###6.0 +- 1.1###5.3 +- 1.2###5.1 +- 0.9

MSM14###4.2 +- 0.9###3.9 +- 0.7###3.3 +- 0.3###3.3 +- 0.9

MSM15###3.7 +- 0.5###3.3 +- 0.3###3.5 +- 0.3###2.9 +- 0.4

Table 7. Assessment of various combinations of IBA on number of roots per shoot and average root length (cm) in groundnut.

Root###GOLDEN###BARI 2001

induction###No. of shoots###No. of roots###Root length###No. of shoots###No. of roots###Root length

media###producing roots (%)###per shoot###(cm)###producing roots (%)###per shoot###(cm)

RIM1###15.5 +- 2.3###0.0 +- 0.0###0.0 +- 0.0###11.3 +- 2.3###0.0 +- 0.0###0.0 +- 0.0

RIM2###23.3 +- 3.0###0.8 +- 0.3###2.0 +- 0.4###17.5 +- 2.0###0.6 +- 0.3###1.7 +- 0.4

RIM3###35.4 +- 4.7###3.1 +- 0.5###2.8 +- 0.7###26.8 +- 3.3###2.3 +- 0.7###2.0 +- 0.6

RIM4###46.7 +- 5.1###1.3 +- 0.3###2.1 +- 0.6###38.4 +- 3.3###1.3 +- 0.5###1.6 +- 0.6

RIM5###39.3 +- 4.1###0.8 +- 0.3###1.7 +- 0.3###27.3 +- 2.9###0.6 +- 0.3###1.3 +- 0.3

RIM6###50.4 +- 3.8###4.4 +- 0.7###2.0 +- 0.6###42.6 +- 4.6###2.4 +- 0.8###1.3 +- 0.3

RIM7###58.8 +- 3.0###6.3 +- 1.1###5.8 +- 0.8###50.7 +- 5.1###4.2 +- 1.0###3.6 +- 0.8

RIM8###90.5 +- 5.8###8.3 +- 1.4###8.1 +- 1.5###76.3 +- 5.8###6.3 +- 1.3###7.0 +- 1.1

RIM9###79.6 +- 5.5###4.8 +- 0.8###6.3 +- 1.3###70.8 +- 4.8###3.4 +- 0.9###4.3 +- 0.7

RIM10###76.3 +- 4.6###3.2 +- 0.3###4.7 +- 0.8###66.6 +- 4.4###2.1 +- 0.3###2.7 +- 0.8

RESULTS

Morphogenetic response of explants on callus induction media: In order to obtain callus cultures, embryo slices were placed horizontally in contact with the surface of callus induction media (CIM) having various combination of PGRs (Table 4) (Figure a). After 10 days of culturing, explants expanded five times in their original size (Figure b) and turned either brown and compact (Golden) (Figure c) or yellow and friable (BARI 2001) (Figure d). In the first experiment, response of explants was checked by placing embryo slices on MS media supplemented only with different levels of BAP (Table 4), where the callus induction frequency (CIF) was no more than 19% and 13% in Golden and BARI 2001, respectively. In order to enhance callus induction frequency different levels of PGRs (BAP + IAA and BAP + NAA) on MS media were used (Table 4). Optimum CIF (86% and 78%) was obtained on CIM 13 in these two varieties (Table 4). No any calli formed in control.

Furthermore, at the higher levels of BAP and NAA (CIM 10) callus inhibition was seen in both varieties. BAP in combination with TDZ and KIN promotes multiple shoot induction: From the results it has been confirmed that regeneration of multiple shoots attained when healthy callus was transferred on multiple shoot regeneration media (MSM) with reduced levels of hormones. BAP alone as well as in combination with TDZ and KIN was used to facilitate the multiple shoot induction. The brownish and dead parts of calli were removed with sharp, sterilized scalpel and sub-cultured on MSM media that have PGRs (Table 5) for 15 days. The calli swelled radially from the excised portion and prolonged the apical meristem five times more than the calli placed on simple MS media (devoid of hormone). After 15 days of explants on MSM, approximately 10-20 buds per explant were formed. In control experiment (hormone-free medium) no bud formation occurred.

The results showed significant variation in number of multiple shoots/plant between the varieties (Table 5). The explants on MSM1-MSM5 were unable to induce maximum multiple shoots in both the varieties (Table 5). MSM9 facilitated multiple shoot induction (4.6 and 4.1) with considerable shoot length (4.8 and 4.3 cm) and number of leaves/shoot (4.4 and 4.1) (Table 6) as compared to MS media devoid of hormone and MSM1-MSM5 in Golden and BARI 2001. The most effective combination of these PGRs was observed on MSM13 media producing highest number of multiple shoots/plant (9.0 and 6.6) with highest shoot length (6.0 and 5.1 cm) and maximum number of leaves/shoot (6.1 and 5.3) in Golden and BARI 2001, respectively as shown in Table 5 and Table 6. The MSM10 and MSM15 having higher concentrations of hormones inhibited the multiple shoot formation, average shoot length and leaves/explant forming the calli on the lower surface of shoots in both the varieties (Figure f).

After the excision of older shoots, new shoots were being produced on the clump by placing on fresh MSM. The buds and shoots produced on older clumps were sub cultured on shoot elongation media (SEM) (data not shown) after every two weeks and transferred on MSM for multiple shoots regeneration.

Root induction needs no PGR application: On average of 2-4 cm elongated shoots were detached of multiple shoots bunch formed from callus that has been shifted to RIM. Substantial rooting system was seen on MS media fortified with IBA concentrations (autoclaved and filter sterilized). After 2-3 weeks, regenerated shoots produced roots via callus induction in both varieties (Figure g). The shoots on RIM1-RIM5 did not promote rooting (Table 7). The Highest rooting efficiency was achieved in Golden variety on RIM8, where 90% of the regenerated shoots produced roots when cultured medium exhibiting maximum root length (8.1 cm) and maximum number of roots/plants (8.3) (Table 7). In variety BARI 2001 there were a minute response to rooted plants (76%) with smaller root length (7.0 cm) and reduced number (6.3) of roots/plant compared to Golden (Table 7).

Roots on RIM8 were healthy and normal in appearance, whereas the use of IBA (>1.5 mg/L) lead to inhibit rooting system calli formation on the basal portion of plantlets resulting in the thickness and shortening of roots (Figure h). then these plants were shifted to hydroponic condition having yoshida solution for the elongation of roots. After 2 weeks transferring of explants on hydroponics system, significant root development occurred in both the varieties. The plantlets were effectively adapted in glasshouse for a week and then shifted in pots containing 1:3 manure and sand with 85% survival rate. These plants appeared uniform, healthy, and morphologically com-parable to the donor plants (Figure. i).

For in vitro root regeneration individual shoots were detached from the group of multiple shoots and moved to MS media having IBA (0.5-2.5 mg/l) for 2 weeks. It was evaluated from this study that there is no need of phytohormones for root formation (Asylin-Ozudogru et al., 2005). About 2-5 cm shoots were shifted to root induction media and later on after 3-4 weeks, root formation started in both the varieties. It is already reported that various doses of IBA, optimum root induction was seen at 1.5 mg/l IBA (Perveen et al., 2011), giving the highest root induction frequency with maximum root length in both the varieties. At 1.5 mg/l IBA healthy roots were appeared on the base of shoots while, at higher levels of IBA (> 1.5 mg/l) roots become thick and shorten due to the formation of callus, resulting in inhibition of roots formation.

After abundant formation of roots, these plantlets were shifted to hydroponics culture in Yoshida solution (Yoshida et al. 1976) for 1-2 weeks and after every 4 days fresh Yoshida solution was added. When significant root elongation with lateral roots developed, then these plants were acclimatized in plastic bags. After some time (2 weeks) these acclimatized plants were again transferred to soil pots to maintain and grow in new environmental condition. Similar results were also reported by Venkatachalam et al. (1997); Banerjee et al. (2007); Verma et al. (2009) by using mixture of hormones i.e. IBA, NAA and KIN for root induction in groundnut. Present technique provides a reliable and high frequency yielding process for obtaining morphologically normal peanut plants in a short time.

Conclusion: The present study provides a reliable and high frequency yielding process for obtaining morphologically normal peanut plants in a short time. The best hormonal combination was BAP along with NAA resulting in the highest callus induction frequency. The highest CIF was noted with 5.5 mg/l BAP and 1.5 mg/l NAA in Golden, compared to other combinations. BAP in combination with TDZ and KIN promoted multiple shoot induction. MS media having 5.5 mg/l BAP and 1.5 mg/l NAA was found to be best by producing the highest callus induction frequencies (86 and 78%) in Golden and BARI 2001, respectively. Similarly, the maximum multiple shoots/plant (9 and 6) with optimum length of shoots (4.8 and 4.1 cm) was obtained with the application of 4 mg/l BAP along with 1 mg/l NAA and 1.1 mg/l TDZ in Golden and BARI 2001, respectively. Roots on RIM8 [MS + IBA (1.5 mg/l)] were healthy and normal in appearance, whereas the use of IBA (>1.5 mg/L) led to inhibit the rooting system.

Acknowledgements: The authors highly acknowledge National Institute for Genomics and Advanced Biotechnology (NIGAB), NARC, Islamabad.

REFERENCES

Abdellatef, E., and M. M. Khalafallah (2007). Adventitious shoot formation and plant regeneration in medium staple cotton (Gossypium hirsitum L.) cultivar (Barac B-67). Int. J. Agri. Biol. 9(6): 913-916.

Akasaka, Y., H. Daimon, and M. Mii (2000). Improved plant regeneration from cultured leaf segments in peanut (Arachis hypogaea L.) by limited exposure to thidia-zuron. Plant Sci. 156: 169-175.

Analytical Software. (2005). Statistix version 8.1: User's manual. Analytical Software, Tallahassee, Florida.

Asylin-Ozudogru, E., Y. Ozden-Tokatli, and A. Akcin (2005). Effect of silver nitrate on multiple shoot formation of Virginia-type peanut through shoot tip culture. In Vitro Cell. Dev. Biol. Plant 41: 151-156.

Baker, C. M., R. E. Durham, J. A. Burns, W. A. Parrott, and H. Y. Wetzstein (1995). High frequency somatic embryogenesis in peanut (Arachis hypogaea L.) using mature, dry seed. Plant Cell Rep. 15: 38-42.

Banerjee, P., S. Maity, S. S. Maiti, and N. Banerjee (2007). Influence of genotype on in vitro multiplication potential of Arachis hypogaea L. Acta Bot. Croat. 66: 15-23.

Cheng, M., D. C. H. Hsi, and G. C. Philip (1992). In vitro regeneration of valencia-type peanut (Arachis hypogaea L.) from cultured petiolules, epicotyl sections and other seedling explants. Peanut Sci. 19: 82-87.

Chengalrayan, K., S. Hazra, and M. Gallo-Meagher (2001). Histological analysis of somatic embryogenesis and organogenesis induced from mature zygotic embryo-drived leaflets of peanut (Arachis hypogaea L.). Plant Sci. 161: 415-421.

Eapen, S., and L. George (1993). Plant regeneration from leaf discs of peanut and pigeonpea: Influence of benzyladenine, indoleacetic acid and indoleacetic acid-amino acid conjugates. Plant Cell Tiss. Organ Cult. 35: 223-227.

Feyissa, T., M. Welander, and L. Negash (2005). In vitro regeneration of Hagenia abyssinica (Bruce) J.F. Gmel. (Rosaceae) from leaf explants. Plant Cell Rep. 24: 392-400.

Franklin, C. I., T. N. Trieu, R. A. Gonzales, and R. A. Dixon (1991). Plant regeneration from seedling explants of green bean (Phaseolus vulgaris L.) via organogenesis. Plant Cell Tiss. Org. 24: 199-206.

Hu, C. Y., and P. J. Wang (1983). Meristem, shoot tip and bud cultures. In: Evan DA, Sharp WR, Ammirato PV, Yamada Y (eds) Hand-book of plant cell culture. 1. Macmillam Publ. New York 177-227.

Hutchinson, M. J., J. M. Tsujita, and P. K. Saxena (1994). Callus induction and plant regeneration from mature zygotic embryos of a tetraploid Alstroemeria (A. pelegrinax A. psittacina). Plant Cell Rep. 14: 184 -187.

Ishag, S., G. O. Magdoleen, and M. K. Mutasim (2009). Effects of growth regulators, explant and genotype on shoot regeneration in tomato (Lycopersicon esculentum c.v. Omdurman). Int. J. Sustain. Crop Prod. 4(6): 7-13.

Kaeppler, S. M., F. Heidi, Kaeppler, and R. Yong (2000). Epigenetic aspects of somaclonal variation in plants. Plant Mol. Biol. 43: 179-188.

Khalafalla, M. M., and K. Hattori (2000). Differential in vitro direct shoot regeneration responses in embryo axis and shoot tip explant of faba bean. Breed. Sci. 50: 117-22.

Lacroix, B., Y. Assoumou, and R. S. Sangwan (2003). Efficient in vitro direct shoot organogenesis and regeneration of fertile plants from embryo explants of Bambara groundnut (Vigna subterranea L. Verdc.). Plant Cell Rep. 21: 1153-1158.

Lemaux, P. G. (2008). Genetically engineered plants and foods: a scientist's analysis of the issues (Part I). Annu. Rev. Plant Biol. 59: 771-812.

Cuc, L. M., E. S. Mace, J. H. Crouch, V. D. Quang, T. D. Long, and R. K. Varshney (2008). Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea). BMC Plant Biol. 8: 55.

Lyyra, S., A. Lima, and S. Merkle (2006). In vitro regeneration of Salix nigra from adventitious shoots. Tree Physiol. 26: 969-975.

Mallikarjuna, K., and G. Rajendrudu (2007). High frequency in vitro propagation of Holarrhena antidysenterica from nodal buds of mature tree. Biol. Plant. 51: 525-529.

McKently, A. H., G. A. Moore, and F. P. Gardner (1990). In vitro plant regeneration of peanut. Crop Sci. 30: 192-196.

Moretzsohn, M. C., L. Leoi, K. Proite, P. M. Guimaraes, S. C. M. Leal-Bertioli, M. A. Gimenes, W. S. Martins, J. F. M. Valls, D. Grattapaglia, and J. Bertioli (2005). A microsatellite-based, gene-rich linkage map for the AA genome of Arachis (Fabaceae). Theor. Appl. Genet. 6: 1060-1071.

Mroginski, L. A., K. K. Kartha, and J. P. Shyluk (1981). Regeneration of peanut (Arachis hypogaea) plantlets by in vitro culture of immature leaves. Can. J. Bot. 59: 826-830.

Murashige, T., and F. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15: 473-497.

Palanivel, S., S. Parvathi, and N. Jayabalan (2002). Callus induction and plantlet regeneration from mature cotyledonary segments of groundnut (Arachis hypogaea L.). J. Plant Biol. 45(1): 22-27.

Pasupuleti, J., S. N. Nigam, K. P. Manish, P. Nagesh, and K. V. Rajeev (2013). Groundnut improvement: use of genetic and genomic tools. Front. Plant Sci. 4: 23.

Perveen, S., A. Varshney, M. Anis, and I. M. Aref (2011). Influence of cytokinins, basal media and pH on adventitious shoot regeneration from excised root cultures of Albizia lebbeck. J. Forest Res. 22: 47-52.

Radhakrishnan, T. (1996.) In vitro studies in the genus Arachis. Ph.D. thesis submitted to the University of Saurashtra, Rajkot, India.

Radhakrishnan, T., T. G. K. Murthy, K. Chandran, and A. Bandyopadhyay (2000). Micropropagation in peanut (Arichis hypogaea L.). Biol. Plant 43: 447-450.

Raghu, A. V., S. P. Geetha, G. Martin, I. Balachandran, and P. N. Ravindran (2006). Direct organogenesis from leaf explants of Embelia ribes Burm. - a vulnerable medicinal plant. J. Forest Res. 11: 57 -60.

Robinson, P. J., S. Srivardhini, and G. Sasikumar (2011). Somatic embryogenesis and plant regeneration from cotyledon tissue of Arachis hypogaea L. Res. Plant Biol. 1(3): 21-27.

Seitz, M. H., H. T. Stalker, and C. C. Green (1987). Genetic variation for regenerative response in immature leaflets cultures of the cultivated peanut, Arachis hypogaea. Plant Breeding 98: 104-110.

Shah, S. H., S. Ali, and G. M. Ali (2013). A novel approach for rapid in vitro morphogenesis in tomato (Solanum lycopersicum Mill.) with the application of cobalt chloride. Eur. Acad. Res. 1(9): 2702-2721.

Shah, S. H., S. Ali, S. A. Jan, and G. M. Ali (2014a). Assessment of carbon sources on in vitro shoot regeneration in tomato. Pakistan J. Agri. Sci. 51(1): 197-207.

Shah, S. H., S. Ali, S. A. Jan, J. U. Din, and G. M. Ali (2014b). Assessment of silver nitrate on callus induction and in vitro shoot regeneration in tomato (Solanum lycopersicum Mill.). Pakistan J. Bot. 46(6): 2163-2172.

Shah, S. H., S. Ali, S. A. Jan, J. U. Din, and G. M. Ali (2015). Callus induction, in vitro shoot regeneration and hairy root formation by the assessment of various plant growth regulators in tomato (Solanum lycopersicum Mill.). The J. Anim. Plant Sci. 25(2): 528-538.

Shah, S. H., N. Khan, S. Q. Memon, M. Latif, M. A. Zia, A. Muhammad, K. Nasir, and Zafarullah (2020). Effects of auxins and cytokinins on in vitro multiplication of banana (musa spp.) variety 'W-11' in Pakistan. The J. Anim. Plant Sci. 30(1): 98-106.

Shan, L., T. Guiying, X. Pingli, L. Zhanji, and B. Yuping (2009). High efficiency in vitro plant regeneration from epicotyls explants of Chinese peanut cultivars In Vitro Cell. Dev. Biol.-Plant 45: 525-531.

Sharma, K. K., and V. Anjaiah (2000). An efficient method for the production of transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium tumefaciens-mediated genetic transformation. Plant Sci. 159: 7-19.

Taji, A., P. P. Kumar, and P. Lakshmanan (2002). In vitro Plant Breeding. London, Hayworth Press Inc.

Tiwari, S., D. K. Mishra, A. Singh, P. K. Singh, and R. Tuli (2008). Expression of a synthetic cry1EC gene for resistance against Spodoptera litura in transgenic peanut (Arachis hypogaea L.). Plant Cell Rep. 27: 1017-1025.

Tiwari, S., and R. Tuli (2009). Multiple shoot regeneration in seed-derived immature leaflet explants of peanut (Arachis hypogaea L.). Sci. Hortic. 21: 223-227.

Upadhyaya, H. D., R. Ortiz, J. P. Bramel, and S. Singh (2003). Development of a groundnut core collection using taxonomical, geographical and morphological descriptors. Genet. Resour. Crop Ev. 50(2): 139-148.

Venkatachalam, P., P. B. Kavi Kishor, and N. Jayabalan (1997). High frequency somatic embryogenesis and efficient plant regeneration from hypocotyl explants of groundnut (Arachis hypogaea L.). Curr. Sci. 72: 271-275.

Venkatachalam, P., and N. Jayabalan (1997). Effect of auxins and cytokinins on efficient plant regeneration and multiple-shoot formation from cotyledons and cotyledonary-node explants of groundnut (Arachis hypogaea L.) by in vitro culture technology. Appl. Biochem. Biotech. 67(3): 237-247.

Venkatachalam, P., A. Subramaniampillai, and N. Jayabacan (1996). In vitro callus culture and plant regeneration from different explants of groundnut (Arachis hypoyaea L). Breeding Sci. 46(4): 315-320.

Verma, A., C. P. Malik, V. K. Gupta, and Y. K. Sinsinwar (2009). Response of groundnut varieties to plant growth regulator (BAP) to induce direct organogenesis. World J. Agric. Sci. 5(3): 313-317.

Yoshida, S. I., D. A. Forno, J. H. Cock, and K. A. Gomez (1976). Laboratory manual for physiological studies of rice. International Rice Research Institute. Manila.
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Author:N. Ahmad, M. R. Khan, S. H. Shah, M. A. Zia, I. Hussain, A. Muhammad and G. M. Ali
Publication:Journal of Animal and Plant Sciences
Geographic Code:9PAKI
Date:Dec 31, 2020
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