Philippine Journal of Science145 (2): 181-187, June 2016ISSN 0031 - 7683Date Received: ?? Feb 20??

Key words: ‘Lakatan’ banana, postharvest traits, shelf life, somaclonal variation, 2,4-D

Potential Banana cv 'Lakatan' Somaclones Inducedby Long Culture Period and High 2,4-D Concentration

University of Southern Mindanao (USM)Kabacan, 9407 Cotabato, Philippines

*Corresponding author: eperezkalaw@gmail.com

Emma K. Sales* and Harem R. Roca

The study was undertaken to determine the effect of long subculture and high dosage of 2,4-D on the yield and other postharvest traits of banana cv ‘Lakatan’ somaclones. Morphological evaluation was done on 2,040 plants (planted in a 2.5ha field (3x3m distance of planting, laid in Randomized Complete Block Design (RCBD) in factorial arrangement) using the International Network for the Improvement of Banana and Plantain (INIBAP) postharvest evaluation procedure. Out of these 2,040 plants, 40 somaclones were selected based on their better performance compared to the untreated plants(control). Results showed that prolonged subculture and addition of high concentration of 2,4-D produced both positive and negative variations. Positive variation was exhibited by heavier bunch weight, earlier flowering, longer shelf life and a larger number of hands, which translate into increased income. Negative variation, on the other hand, included dwarfism, delayed flowering and a lesser number of hands.

INTRODUCTION

In-vitro culture is a popular method of vegetatively propagating crop plants with high commercial value. Ideally, all plants generated from tissue culture are true-to-type i.e. have identical constitution from that of the mother or original plants. However, contrary to this, high incidence of off-types is a major concern. This variation is called somaclonal variation and may be defined as genetic and phenotypic variation among clonally propagated plants of a single donor clone (Kaeppler et al. 2000). Larkin & Scowcroft (1981) pointed out that a consistent proportion of the regenerated plants differ from the original parental type when submitted to tissue-culture techniques. Methods for detection of somaclonal variation have been explored for many years (Noval 1980). Although such variation may provide a useful source of genetic variability for crop improvement, it is undesirable in plant propagation. Moreover, Sultana et al. (2005)

who worked on Indica Basmati rice, observed that tissue culture generates a wide range of variation ranging from 35.3% - 45.2%, which is related to the incubation time and is cultivar specific. Also, Smith (1998) observed that variation in plants derived from shoot tip culture can vary from 0-70% depending on the genotype. Therefore, this genetic instability can be a risk in germplasm handling and storage; however, it can also provide another source of novel and useful variability (Vuylsteke 1998) especially in crops where conventional breeding is difficult. Ehsanpour et al. (2007) observed that somaclonal variation was useful in selecting callus formation for desirable traits such as salt and drought tolerance or secondary metabolite production.

In Cavendish banana, somaclonal varaiation is associated with long duration in tissue culture and from a long period of the multiplication phase of meristem culture (Khayat et al. 2004). In another study, Skirvin et al. (1994) observed that somaclonal variation was related to growth regulator, cultivar variability, duration of in vitro culture, ploidy level and explant source. Furthermore, certain chemicals

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like 2,4-D also enhance somaclonal variation (Sultana et al. 2005).

According to Boranayaka et al. (2010), induced mutation is an important tool to create desired characteristics which are otherwise found in extensive germplasm collections. Moreover, variation resulting from micropropagation i.e. somaclonal variation, appears to be frequent in Musa such that somaclonal variation can be an effective tool in the improvement of horticultural traits of banana (Tang 2005). Larkin & Scowcroft (1981) suggested that somaclonal variation can be a useful source of novel variation for plant improvement. Similarly, Lestari (2006) reported that somaclonal variation can be an alternative technology in plant improvement which is expected to support conventional breeding. Through somaclonal variation desirable traits such as larger fruit size, more interesting flowers (in ornamentals), fruit texture and increased yield (Witjaksono 2003) may be obtained.

MATERIALS AND METHODS

Plant MaterialsCorms of banana cv ‘Lakatan’ were obtained from a plantation in Magpet, North Cotabato, Philippines and were used for in-vitro propagation.. Shoot tip cultures derived from ‘Lakatan’ corms were established using standard tissue culture procedures for disinfection, culture initiation and maintenance (Hwang & Ko 1990).

Shoot tips were inoculated on full strength Murashige & Skoog (MS)(1962) medium solidified with 7% agar supplemented with various levels of 2,4-D (3, 6, 9, 12 µM/L, respectively). Cultures were maintained at 27 0C with 24 h white light provided by a 40 watt fluorescent lamp.

After 21 days, the initial cultures were further subdivided into 2-3 sections. Every 21 days thereafter, the cultures were transferred to a fresh medium. The cultures were maintained until the 15th subculture to determine the effect of age of culture on the induction of somaclonal variation in-vitro. Beginning on the third cycle until the 15th cycle, observations and data on the following was gathered: number of days from planting to flowering, number of days from flowering to harvest, number of days to 50% yellowing, number of days end of shelf life, bunch weight, average weight (fruit and hand), number of hands and fingers, fruit length, fruit girth, TSS (%Brix), pH, and percent dry matter content of fruit at harvest were recorded. Plantlets derived per treatment combination were planted in the greenhouse for four weeks.

The in-vitro derived plantlets were planted in a 2.5

hectareplot following a 3 x 3 m distance of planting and laid out in a Randomized Complete Block Design (RCBD) in factorial arrangement with 30 plants per somaclone (as replications). Field performance based on agronomic, pre-harvest (e.g. days from planting to flowering and days from flowering to harvesting) and postharvest parameters and shelf life were observed and recorded. Comparison observed through the performance of somaclones with different treatment combinations with that of the control or untreated sample plants using T-test and LSD was done.

Evaluation at the nursery level was also done by comparing the treated samples with the control. Candidate plants showing putative off-types were selected and used for further analysis.

RESULTS AND DISCUSSIONThe effect of the various 2,4-D concentrations on the number of days to shoot formation, number of shoots formed previously established in MS culture media is shown in Table 1. Culture media supplemented with 9 µM 2,4-D were the earliest (49 days) to form shoots after initial subculture. In contrast, culture samples supplemented with 12 µM 2,4-D formed shoot 52 days after initial subculture. On the other hand, cultures supplemented with 3 and 6 µM 2,4-D (51 and 51.3 days, respectively) did not differ significantly with those without 2,4-D supplementations (50 days).

Table 1. Effects of 2,4-D concentrations on the number of days to shoot formation.

Days to shoot formation

No. of shoots produced after 50 days

0 2,4-D 50.2ab 32a

3 uM 2,4-D 51ab 6c

6 uM 2,4-D 51.3ab 7c

9 uM 2,4-D 49.7b 18b

12 uM 2,4-D 52a 5c

*CV 15.5%

In terms of number of shoots formed after 50 days, addition of 2,4-D significantly decreased the number of shoots produced. For example, shoot tips inoculated unto MS media without 2,4-D produced 32 shoots, while those shoot tips inoculated onto various 2,4-D concentrations (3, 6, 9 and 12 µM 2,4-D, respectively) had significantly lower shoots produced (6, 7, 18, 5, respectively).

The above results implied that high 2,4-D concentration as a supplement to culture media had negative effects on the shoot initiation and multiplication of in-vitro culture bananas.

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However, Iqbal et al. (2011) observed that 3 mg/L 2,4-D is very efficient in inducing embryonic callus with desirable quality friable callus in peanut. Likewise, El-Sayed et al. (2012) reported that 2,4-D and other chemicals can induce mutation in banana plants.

Multiplication rate of shoots from subculture 3 to subculture 15 in relation to the various 2,4-D concentration is shown in Figure 1. In general, a double sigmoid curve pattern of multiplication rate was observed. Initially, there was slow increase from subculture 3, to subculture 5 and a rapid increase from subculture 5 until subculture 9, then a rapid decrease from subculture 10 until subculture 11 and a sigmoid pattern from subculture 3 to subculture 9. All the other treatments followed the same trend. This is expected since multiplication rate in in-vitro culture follows an exponential increase, hence, this is a popular technique in mass producing vegetatively propagated plants e.g. banana (Akin-Idowu et al. 2009, Bhosale et al. 2011). Muhammad et al. (2004) reported that the maximum number of shoots was produced from each shoot tip after five subcultures.

Postharvest performance of 40 selected ‘Lakatan’ somaclonesThe list of all potential somaclones and their corresponding beneficial traits is presented in Table 2. Criteria for selection were based on superior performances (both at harvest and postharvest) exhibited by the individual somaclones over that of the control. In terms of number of days from flowering to harvest, somaclone # 19 from cycle 10 treated with 12 µM 2,4-D (C10T5) was harvested 56 days after flowering. It means that it matured 32 days earlier than the control (88 days). This was followed by somaclone # 21 treated with 12 µM 2,4-D, subcultured

Figure 1. Multiplication rate of banana shoot tips in response to various 2,4-D concentrations.

until the 10th cycle which was harvested 65 days after flowering showing an earlier maturity of 23 days over the control.

Another important trait where significant variation was observed is in terms of bunch weight, where 19 somaclones were significantly heavier than the control. The heaviest bunch weight (20.78 kg) was observed from somaclone # 31 from cycle 11 without 2,4-D. This means a 7.78 kg heavier bunch than the control (12.10 kg), and 5.78 kg heavier than the usual normal Philippine ‘Lakatan’ which bears an average weight of 13.5 – 20.28 kg. These results indicate that prolonged subculture might be a factor that can induce the said beneficial or positive traits . It can be noted that somaclone # 20 derived from cycle 9 treated with 9 µM 2,4-D gave lower bunch weight, however, it yielded a higher number of fingers (20 fingers/hand) and the highest value of 32.20 Brix compared to the control which had an average of 16 fingers per hand and 260 Brix TSS. For the number of hands per bunch, seven somaclones were observed to have nine hands/bunch which was one hand/bunch more than the control (8 hands/bunch).

All of these seven somaclones were subjected to a prolonged subculture of 11, 12 and 13 cycles/subcultures but lower 2,4-D concentration. In terms of the number of fingers per hand, 29 somaclones gave one to five more fingers compared to the control which had 16 fingers per hand. These 29 somaclones had fingers ranging from 17-21 per hand. Furthermore, somaclone # 48 from cycle 14 treated with 12 µM 2,4-D took the longest (16 days) to 50% yellowing. This was nine days longer than the control which can be an indication of longer shelf life. Furthermore, 28 somaclones gave a range of 8-15 days

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Figure 2. Different somaclones of Banana cv ‘Lakatan’ with higher yield performance.

from green to 50% yellowing. Somaclone # 17 from cycle 12 treated with 9 µM 2,4-D (C12T4 # 17) and somaclone # 48 from cycle 14 treated with 12 µM 2,4-D had 20 days shelf life which was five days longer than the control (15days). Another trait includes pH. It was observed that somaclone # 13 from cycle 13 treated with 9 µM 2,4-D/L showed a pH of 6.78 indicating that the fruit is not acidic. This might be interesting in later studies for nutrient/pharmaceutical consideration/properties.

Other somaclones had pH ranging from 4.43 to 5.57 while that of the control was 4.98. This might be an important indication of the nutritional quality of such somaclone. The results conformed to observation that tissue culture conditions can induce varied amount of genetic changes in different regenerated plants (Soniya et al. 2001). Overall observation showed that prolonged subculture and addition of high dose of 2,4-D can induce somaclonal variation. These findings conform to the results of Fukui (1983) that culture period itself is a mutagenic agent and that these mutations are not simply pre-existing in the ex-plant source. Lee & Philips (1987) and Zehr et al. (1987) stated that there is an “age effect” i.e. the longer the cultures are maintained, the higher the frequency of mutations found in the regenerated plants. Moreover, Mendez et al. (1996) reported that somaclonal variation

can be due to the permanence of culture in-vitro or number of subcultures. Likewise, as previously stated, the presence of certain chemicals like 2,4-D also enhances the rate of somaclonal variation (Sultana et al. 2005).

Table 3 shows the summary of the selected somaclones and their performance. Somaclone # 31 from cycle 11 untreated with 2,4-D (C11T1 # 31) had heavier bunch (20.78 kg) than the control (12.10). In addition, somaclone # 5 from cycle 7 untreated with 2,4-D (C7T1 # 5), and somaclone # 42 from cycle 12 treated with 3 µM 2,4-D (C12T2 # 42) had heavier fingers (140 g) than the control (80 g). Somaclones # 5 and # 38 from cycle 11 treated with 3 µM 2,4-D had a larger number of fingers (21) per hand over the control (16).

The results of the study showed that indeed variation was induced by the treatments employed. Age (prolonged subculture) and high hormone level (2,4-D) were the factors that contributed to the positive and negative traits in somaclones. These results were in agreement with Roux (2004) indicating that the use of in-vitro mutagenesis and tissue culture have been found to make induction and selection of induced somatic mutations more effective. According to Novak et al. (1990), the frequency of phenotypic and morphological variations (plant height,

Sales EK et al.: Potential 'Lakatan' SomaclonesPhilippine Journal of ScienceVol. 145 No. 2, June 2015

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Table 2. Morphological traits of selected banana cv ‘Lakatan’ som

aclones from various treatm

ent combinations (subculture and 2,4-D

levels).

Somaclones

Days from

planting to flow

ering

Days from

flow

ering to harvest

Days to 50%

yellow

ingD

ays end of shelf life

Bunch

wt.(kg)

Average weight

Num

ber ofFruitlength(cm

)

Fruitgirth(cm

)

TSS (%

B

rix)pH

Dry m

atter content of fruit at harvest

(%)

Fruit(g)H

and (kg)H

andsFingers

Group 1

C5T1 # 24 (C

ontrol)220

887

1512.10

801.25

816

12.7810.04

26.04.89

18.30C

5T5 # 23224

914

1011.20

1101.38

713

12.9010.10

284.72

19.30C

6T5#03353

746

1013.50

1051.77

717

16.5310.41

284.56

25.40C

14T3#21287

8510

1517.00

1201.43

816

18.2511.87

25.84.97

21.62C

14T2#34296

928

1516.10

1152.0

818

17.2511.64

24.85.01

18.75G

roup 2C

7T1 # 18318

9411

1417.20

1211.81

816

18.1011.77

25.24.57

19.18C

7T1 # 5334

805

916.20

1402.03

718

18.0411.32

22.84.96

20.44C

13T4#13338

889

1313.50

1101.45

714

17.6011.47

25.06.78

22.03G

roup 3C

7T1 # 27307

858

1317.00

1302.15

716

17.5011.19

23.64.54

19.36C

7T3 # 5332

868

1316.42

1001.79

820

13.9011.21

25.04.87

23.62G

roup 4C

8T1#25318

878

1516.80

1031.79

718

18.7511.54

22.24.17

20.58C

9T4#20372

868

139.40

791.58

620

14.539.78

31.24.89

22.25C

10T5#21359

659

1414.60

981.53

719

17.5310.81

29.05.04

25.20C

10T5#19446

569

1412.00

801.27

818

15.599.88

24.05.17

25.20C

11T2#15277

869

1318.63

1001.54

921

15.2210.11

21.64.73

25.01C

11T2#38293

854

1117.60

1001.73

921

18.010.70

29.24.87

21.50C

11T2#4299

816

1016.90

991.61

918

15.9610.42

26.45.20

21.60C

12T4#17314

8413

2016.50

1201.83

818

17.3310.65

26.64.73

22.52C

12T1#28350

8414

1816.25

1001.64

919

16.4810.74

27.04.79

18.26C

12T3#33341

8610

1515.90

1202.09

714

17.2911.08

27.05.09

22.51C

12T2#42328

924

1113.00

1401.59

716

17.2910.40

24.24.95

24.08C

13T2#1374

8611

1619.15

1001.30

919

17.011.35

25.05.06

22.50C

13T2#31346

9110

1517.80

1051.79

918

15.8510.12

24.04.79

18.82C

13T2#19330

8610

1817.00

801.54

720

15.2010.97

24.24.75

23.50C

13T2#24436

827

145.00

60.60

614

11.5010.50

26.24.78

20.46G

roup 5C

7T3 # 21307

8811

1516.69

1251.53

819

15.599.87

23.04.67

22.57C

7T5 # 23282

857

1315.65

1001.64

818

16.0010.72

30.04.78

22.54C

10T2#21360

629

2114.10

1211.71

716

17.1010.89

26.25.20

23.34C

11T1#31438

899

1520.78

1201.90

919

18.1011.80

24.04.70

22.69C

11T2#9298

7812

1716.90

951.90

820

17.8010.68

23.85.27

22.50G

roup 6C

13T2#30316

8615

1916.25

901.64

818

16.6510.84

28.24.93

22.01G

roup 7C

13T2#41437

818

167.45

80.90

714

14.011.50

26.04.65

22.08U

SMA

RC

--

--

11.2292.6

1.587

1712.68

9.1222.78

--

Group 8

C7T4#25

30884

914

15.43110

2.208

2015.50

10.2824.0

4.7521.81

Group 9

C8T1#7

37983

1518

11.3095

1.327

1715.05

9.8128.0

4.5122.34

C14T3#43

37781

1116

13.20120

1.647

1618.50

11.5025.6

4.4321.17

C14T2#31

33087

711

11.7080

1.506

1816.0

10.9829.9

4.8621.75

Group 10

C14T5#48

31779

1620

9.8580

1.236

1716.50

10.025.0

4.9917.45

C15T2#35

33288

1014

17.00122

2.087

1715.05

11.3525.0

4.6719.82

C15T3#47

30787

1519

15.10115

1.787

1915.25

10.4527.2

5.0221.30

C= cycle

T2 =3µM

2,4-D

T4 = 9µM 2,4-D

T1= 0 2,4-D

T3 = 6µM 2,4-D

5 = 12µM

2,4-D*Statistical A

nalysis – t-Test A

ll data are significantly different from that of the control at 5%

level (T-test)

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Table 3. Summary of the selected somaclones and the corresponding traits exhibited expressed in response to prolonged subcultures and 2,4-D level.

Somaclone Heavier Bunch (kg) Somaclone Weight of finger (g) Somaclone Number of fingers/hand

C11T1#31 20.78 C7T1#5 140 C11T2#15 21

C7T1#18 17.20 C12T2#42 140 C11T2#38 21

C7T1#27 17.00 C10T2#21 121 C7T3#5 20

C11T2#15 18.63 C11T1#31 120 C7T4#25 20

C13T2#1 19.15 C12T4#17 120 C11T2#9 20

C13T2#31 17.80 C14T3#21 120 C13T2#19 20

C13T2#19 17.00 C14T3#43 120 control 16

C14T3#21 17.00 control 80

control 12.10

leaf color), physiological variations (growth and sucker multiplication, duration of flowering, fruit ripening), and agronomic variations (bunch qualities) varied from 3 to 40% in the first generation, depending on the genotype. From a practical point of view, it is most important that off-types (whether positive or negative) can be identified at an early stage. For positive variation, farmers are assured of more benefits especially on yield translated into income while for negative variations, these can be culled out at the early stage to avoid loss in investments.

CONCLUSIONS AND RECOMMENDATION With the foregoing obtained results, mutation induction through long culture period and high 2,4-D concentration may lead to enhanced heritable variation, hence, there is a need for further testing of advanced generations. This might be useful for functional genomics or breeding applications. This can be exploited for banana postharvest improvement. In addition, yield traits such as bunch weight can be further studied to develop new strains which will give better yield. Further testing of the selected mutant lines for the traits considered is also essential.

ACKNOWLEDGMENTThe authors are very grateful to the Department of Science and Technology (DOST) through Secretary Mario G. Montejo, for the funding support; PCAARRD through Drs. Patricio S. Faylon, Danilo C. Cardenas, and Crops Research Division for facilitating the implementation and completion of this research project; and USM through President Jesus Antonio G. Derije, for the use of facilities and administrative support throughout the conduct of this

project.

LITERATURE CITEDAKIN-IDOWU PE, IBITOYE DO, ADEMOYEGUN

OT. 2009. Tissue Culture as a Plant Production Technique for Horticultural Crops. African Journal of Biotechnology 8(16):3782-3788.

BORANAYAKA MB, IBRAHIM SM, ANANDAKUMAR CR, RAJAVEL DS. 2010. Induced Macro-mutational Spectrum and Frequency in Sesame (Sesamum indicum L.). Indian Journal of Genetics and Plant Breeding 70(2) 155-164

BHOSALE UP, DUBHASHI SV, MALI NS, RATHOD HP. 2011. In vitro Shoot Multiplication in Different Species of Banana. Asian Journal of Plant Science and Research 1(3):23-27.

El-SAYED, EMAN H, SHERIN A, MAHFOUZE AD, SHALTOUT AD, EL-DOUGDOUG KhA, SAYED RA. 2012. Chemical Mutation Induction In vitro Cultured Shoot Tip of Banana CV Grand Nain for Resistance to Some Virus Diseases. International Journal of Virulogy 8 (2):178-190.

EHSANPOUR AA, MADANI S, HOSEINI M. 2007. Detection of Somaclonal Variation in Potato Callus Induced by UV-C Radiation using RAPD, Plant Physiology 33(1-2), pp 3-11.

FUKUI K. 1983. Sequential Occurrence of Mutations in a Growing Rice Callus. Theoretical and Applied Genetics 65:225-230.

HWANG SC & KO WH. 1990. Selection of Improved Cavendish Banana Mutants Resistant to Race-4 of Fusarium oxysporum f.sp. cubense. Acta Horticulturae 275:417-423.

Sales EK et al.: Potential 'Lakatan' SomaclonesPhilippine Journal of ScienceVol. 145 No. 2, June 2015

186

IQBAL M, NAZIR F, IQBAL J, TEHRIM S, ZAFARI Y. 2011. In vitro Micropropagation of Peanut (Arachis hypogea) through Direct Somatic Embryogenesis and Callus Culture. Journal of Agriculture and Biology 13:811-814.

KAEPPLER SM, KAEPPLER HF, RHEE Y. 2000. In: Epigenetic Aspects of Somaclonal Variation in Plants. Plant Molecular Biology Report 43:179-188.

K H AYAT E , D U V D E VA N I A , L A H AV E , BALLESTEROS BA. 2004. Somaclonal Variation in Banana (Musa acuminate cv Grande Naine). Genetic Mechanism, Frequency, and Application as a Tool for Clonal Selection. In:Banana Improvement: Cellular, Molecular Biology and Induced Mutations, Jain, S.M. and R. Swennen (Eds.). Science Publishers, Enfield, USA, pp:97-109.

LARKIN PJ, & SCOWCROFT WR. 1981. Somaclonal Variation - a Novel Source of Variability from Plant Cell Cultures for Plant Improvement. Theoretical and Applied Genetics, Berlin, 60, p.197-214.

LEE ML, & PHILLIPS RL. 1987. Genetic Variability in Progeny of Regenerated Maize (Zea mays L.) Plants. Genome 29:344-355.

LESTARI EG. 2006. Hubungan Antara Kerapatan Stomata Dengan Ketahanan Kekeringan Pada Somaklon Padi Gajahmungkur, Towuti,dan IR 64. Biodiversitas 7 91):44-48.

MENDEZ BMJ, MENDEZ FJ, NETO AT, DEMETRIO CGB, PUSKE OR. 1996. Efficacy of Banana Plantlet Production by Micropropagation. Pesqui Agropecu Bras., 31:863-867.

MURASHIGE T, SKOOG FA. 1962. A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures. Physiologia Plantarum, 15, p. 473-497.

MUHAMMAD A, HUSSAIN I, NAQUI SM, RASHID H. 2004. Banana Plantlet Production through Tissue Culture. Pakistan Journal of Botany 36(3):617-620.

NOVAK FJ, AFZA R, DUREN MV, OMAR MS. 1990. Mutation Induction by Gamma Irradiation of In vitro Cultured Shoot-tips of Banana and Plantain (Musa sp.). Tropical Agriculture 67:21-28.

NOVAL F. 1980. Phenotype and Cytological Status of Plants Regenerated from Callus Cultures of Allium sativum. L. Z. Pflanzenzeucht 84:280.

ROUX NS. 2004. Mutation Induction in Musa-Review. In:Banana Improvement: Cellular, Molecular Biology and Induced Mutations, Jain, S.M. and R. Swennen (Eds.). Science Publishers, Enfield, USA. pp:23-32.

SKIRVIN RM, MCPHEETERS KD, NORTEN M. 1994. Source and Frequency of Somaclonal Variation. Horticultural Science 29: 1232-1237.

SMITH MK. 1998. A Review of Factors Influencing the Genetic Stability of Micropropagated Bananas. Fruits 43 (4):219-223.

SONIYA EV, BANERYEE NS, DAS MR. 2001. Genetic Analysis of Somaclonal Variation among Callus Derived Plants of Tomato. Current Science 80(9): 1213-1215.

SULTANA R, TAHIRA F, TAYYAB H, KHURRAM B, SHIEKH R. 2005. RAPD Characterization of Somaclonal Variation in Indica basmati Rice. Pakistan Journal of Botany 37(2):249-262.

TANG CY. 2005. Somaclonal Variation: A Tool for the Improvement of Cavendish Banana Cultivars. Proc. 11 and IS on Biotech of Trop. and Sub-tropical species. Eds. W.C. Chang and R. Dew. Acta Horticulturae. 692. International society for Horticultural Science 2005 pp 61-65.

VUYLSTEKE D. 1998. Field Performance of Banana Micropropagules and Somaclones. In: Somaclonal Variation and Induced Mutation in Crop Improvement. S.M. Jain, D.S. Brar and B.S. Ahloowalia.Dordrecht Kluwer Academic Publishers. Pp. 219-231.

WITJAKSONO. 2003. Peran Bioteknologi Dalam Pemuliaan Tanaman Buah Tropika. Seminar Nasional Peran Bioteknologi dalam Pengenbangan Buah Tropika, IPB. Bogor, 9 Mei 2003.

ZEHR BE, WILLIAMS ME, DUNCAN DR. 1987. Somaclonal Variation among the Progeny of Plants Regenerated from Callus Cultures of Seven Inbred Lines of Maize. Canadian Journal of Botany 61:491-499.

Sales EK et al.: Potential 'Lakatan' SomaclonesPhilippine Journal of ScienceVol. 145 No. 2, June 2015

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