BIOMASS PRODUCTION IN MALE STERILE AND FERTILE GENOTYPES OF CAPSICUM ANNUM L.

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1 Research Article Received: 28 Aug 2013, Accepted: 12 Sept 2013 ISSN: Int. J. Pharm. Biosci. Technol. To cite this Article: Click here International Journal of Pharma Bioscience and Technology. 2013; 1(3): Journal home page: BIOMASS PRODUCTION IN MALE STERILE AND FERTILE GENOTYPES OF CAPSICUM ANNUM L. Mohini K. Suryawanshi, Baig M.M.V. * Department of Botany and Biotechnology, Yeshwant Mahavidyalaya, Nanded , Maharashtra, India. Corresponding Author* address- mmvb@indiatimes.com ABSTRACT: In vitro study was conducted with different genotypes of Capsicum annum L. viz. cytoplasmic male sterile genotype (CMS), genetic male sterile genotypes (GMS), CMS-based maintainer genotype, GMS-based F1 hybrid and CMS-based F1 hybrid of chilli (Capsicum annum L ) for study of biomass production under two different tissue culture experiments, wherein all these genotypes were grown on half-strength MS basal medium which was supplemented with 3% sucrose and 0.01mg/Litre 6-benzyl aminopurine (BAP) alone. Different parameters were studied for calculation of biomass production. In vivo biomass production in CMS/GMS male sterile genotypes of Capsicum annum L. is always more as compared to fertility restorer/fertile genotypes and similar results have been observed for in vitro growth of cytoplasmic male sterile genotype (CMS), genetic male sterile genotypes (GMS) and fertility restorer/fertile genotypes of Capsicum annum L. Therefore, use of tissue culture techniques for production of biomass can be used as a method of co-relating in vivo results as biomass production character plays a significant role in the final fruit setting and fruit production in terms of yield of the plant. The presented study established that in vitro biomass production was maximum in cytoplasmic male sterile genotypes (CMS) and genetic male sterile genotypes (GMS), GMS based F1-hybrid genotypes as compared to other fertility restorer/fertile genotypes of Capsicum annum L. GMS based F1 hybrid was found to produce more biomass just like genetic male sterile genotype indicating the effect of biomass production character in CMS or GMS genotypes in the final fruit setting and fruit production in terms of yield of the plant. Key words: Capsicum annum L., CMS, F1-hybrid, biomass production INTRODUCTION Hot pepper (Capsicum annum L. syn. chilli) is an important commercial crop, cultivated for vegetable, spice, and value-added processed product [1]. Chilies are cultivated for vegetables as well as condiments and also used around the world as sweet peppers, pungent chili peppers, or as a source of dried powders of various colors [2]. Hot pepper (Capsicum annum L. syn. chilli) is an integral part of the spices due to its fruit characteristics such as pungency making fruit production as an important issue during their cultivation. So, fruit yield is always taken into consideration to fulfil the increasing demands of Capsicum fruits in the global market. Utilization of male sterility such as cytoplasmic genetic male sterility (CMS) and nuclear genetic male sterility (GMS) is one of the important approaches for hybrid development in Capsicum annum L. Seed production based on male sterility can achieve high purity and low cost. Although manual pollination is still required, hand emasculation is spared [3]. Cytoplasmic male sterility (CMS) and genetic male sterility (GMS) genotypes have been used in Capsicum annum L. to produce hybrid seed. Hybrid seed production in Capsicum annum L. using cytoplasmic male sterile genotypes (CMS), and genetic male sterile genotypes (GMS) shows significant difference in their biomass production from the stage of seed germination, small plantlet growth stage till mature plant growth stage. This biomass production character is directly related to fruit setting or fruit production in Capsicum crop. So, it is considered as an important character at the time of breeding experiments. Maximum fruit production has become imperative to meet the Pg. 149

2 increasing demand for the fruits of Capsicum annum L. In this research work, in vitro results for biomass production has been studied using tissue culture techniques to co-relate in vivo and in vitro results. Therefore, in the present study, an attempt has been made to study in vitro measurement of biomass production using different sterile (CMS/GMS) and fertile genotypes of Capsicum annum L. which can be useful criterion to choose CMS/GMS genotypes for breeding experiments taking into consideration the effect of biomass production in terms of final fruit setting and fruit production. MATERIALS AND METHODS: The present research study consists of two tissue culture experiments for the study of biomass production of different genotypes. Two different sets of germplasm were used for conducting two experiments. Sources of genetic material used for research work are mentioned in Table 1.One CMS based F1 hybrid (0246*(2+3) and one GMS based F1 hybrid was prepared. A] First tissue culture experiment for biomass production: This experiment uses first set of germplasm which comprises following genotypes: 1. CMS-A (0246-Cytoplasmic Male Sterile Genotype); 2. CMS-B (CMS-Maintainer Genotype); 3. P4-Male /2+3 (Fertility Restorer Genotype); *(2+3)- F1 (Hybrid Genotype); 5. LCA-353 (Fertile Genotype). B] Second tissue culture experiment for biomass production: This experiment uses second set of germplasm which comprises following genotypes: F (Genetic Male Sterile Genotype F (Genetic Male Sterile Genotype); A (Cytoplasmic Male Sterile Genotype); Male (Fertile Genotype); F*(301-Male)- F1 Hybrid Genotype; B (CMS-Maintainer Genotype); 7. LCA-353 (Fertile Genotype). Table 1: Source of genetic material under study. Seeds of Chilli (Capsicum annum L.) plant were used as a germplasm for conducting these two experiments (sources of germplasm are mentioned in Table 1). F1-hybrids were prepared using cytoplasmic male sterile genotype and nuclear genetic male sterile genotype which were used later in these two tissue culture experiments. The seeds were collected and surface sterilized using different sterilizing agents. The seeds were first washed with tap water for 4-5 times to reduce the level of surface organisms and then treated with a mixture of 2% aqueous solution comprising two antiseptics namely chlorhexidine gluconate 0.3% (v/v) and cetrimide 3% (v/v) and 1-2 drops of Twine-20 (surfactant) for 15 minutes. The seeds were later rinsed 3-4 times with distilled water to wash off the detergent solution. This was followed by surface sterilization under aseptic conditions in laminar flow cabinet. The surface sterilization under aseptic conditions started by immersing the seeds in 70% ethanol for 1 minute and transferring the seeds into new sterile conical flask. The seeds were then washed several times with sterile distilled water. The rest of the procedures were carried out in a laminar flow cabinet after turning on bunsen burner. The seeds were then treated with 0.1% (w/v) mercuric chloride (sterilant) for 15 minutes. This was followed by several washes of seeds with sterile distilled water (5-6 washes). The surface-sterilized seeds were transferred to petri plates. Finally, these seeds were inoculated into culture bottles containing half strength MS basal medium [4]. This half strength MS basal medium was supplemented with 3% (w/v) sucrose and 0.01mg/L 6-benzyl aminopurine (BAP) alone. Auxins (IAA, IBA or NAA) or half-strength MS medium have been used for shoot rooting stimulation [5], [6], [7]. Five seeds of each genotype were inoculated into the culture bottle medium. Three replicates were used for each genotype. The culture bottles containing seeds of different genotypes were placed under fluorescent light in a room with controlled temperature (22 ± 2 C). Germinated Pg. 150

3 seedlings were considered for readings. The seed germination and later seedling growth was observed daily, but the final readings of different parameters of plantlets were recorded after 11 weeks of inoculation of seeds in the half strength MS basal medium [4]. Different parameters were studied by taking readings of all three replicates of each genotype and maximum grown plantlets from each replicate of each genotype were used for recording average in case of all studied parameters. The parameters studied were average shoot length, average number of leaves, average root length (including only the primary roots length), average fresh weight of plantlet and average dry weight of plantlet. Parameters studied for both tissue culture experiments for Biomass Production: 1. Average number of days required for germination: The number of days required for germination of seeds from each genotype from the day of inoculation was recorded. The mean value of the data provided the average number of days required for germination of seeds. 2. Average shoot length: Shoot length was recorded and the mean was calculated. The mean value of the data provided the average shoot length of the plantlet. Average shoot length was calculated and recorded in centimetre (cm). 3. Average number of leaves: The number of leaves per plantlet was recorded and the mean was calculated. The mean value of the data provided the average number of leaves grown on shoot of the plantlet. 4. Average root length (including only the primary roots length): Length of primary roots per shoot was recorded and the mean was calculated. The mean value of the data provided the average root length of the primary roots grown on the shoot of the plantlet. Average root length was recorded in centimetre (cm). 5. Average fresh weight of plantlet: Fresh weight of plantlet was recorded and the mean was calculated. The mean value of the data provided the average fresh weight of the plantlet. Average fresh weight was recorded in gram (gm). 6. Average dry weight of plantlet : Plantlets from each genotype were wrapped separately in an aluminium foil and kept at 50⁰C for 24 hours. Dry weight of plantlet was recorded and the mean was calculated. The mean value of the data provided the average dry weight of the plantlet. Average fresh weight was recorded in gram (gm). RESULTS AND DISCUSSION: A] First tissue culture experiment for biomass production: 1. Number of days required for seed germination: No seed germination was observed till 14 days from the date of inoculation of seeds. After 14 days different genotypes started showing germination at different rates and on different days. The first genotype which was observed to be germinated after 14 days from the date of inoculation was the genotype CMS-A (0246) which was followed immediately by germination of second genotype CMS-B (0246). The third genotype which was germinated was 302-B. 2. Average shoot length: Maximum average shoot length was shown to be 4.7cm and 4.1cm in cytoplasmic male sterile genotype-cms-a (0246) and its maintainer genotype (CMS-B) respectively. Less average shoot length shown was 3.0cm in fertile genotype LCA-353 (Table 2). 3. Average number of leaves: Average number of leaves shown was 5 in cytoplasmic male sterile genotype CMS-A (0246), its maintainer genotype (CMS-B) and fertile LCA- 353 genotype. Average number of leaves in 0246*(2+3) - F1 hybrid genotype was observed to be 3. The maximum average number of leaves is observed as 6 in case of P4-Male (2+3) genotype (Table 2). 4. Average root length (including only the primary roots length): Maximum average root length was shown in cytoplasmic male sterile genotype CMS-A (0246) which is 7.8cm. Average root length in CMS-based maintainer genotype (CMS-B) was shown as 4.6cm. Less average root length shown was 3.1cm in 0246*(2+3) - F1 hybrid genotype (Table 2). 5. Average fresh weight of plantlet: Maximum average fresh weight was shown in cytoplasmic male sterile genotype-cms-a (0246) which was 0.10gm. Average fresh weight of CMS-B was observed as 0.08gm and for 0246*(2+3) - F1 hybrid genotype it was observed as 0.07gm (Table 2). 6. Average dry weight of plantlet : Maximum average dry weight was shown in cytoplasmic male sterile genotype CMS-A (0246), and its maintainer genotype (CMS-B) as 0.006gm and 0.005gm respectively. Average dry weight was counted as 0.004gm in case of both fertility restorer genotype P4-Male (2+3) and fertile genotype LCA-353. A very less average dry weight was shown in 0246*(2+3) - F1 hybrid genotype and it was observed as 0.003g (Table 2). Pg. 151

4 Table 2: Observations of different genotypes including cytoplasmic male sterile genotype, CMSmaintainer genotype, restorer genotype, fertile genotype and CMS-based F 1 hybrid after conducting first tissue culture experiment. Sr. Genotype Avg. Avg. Shoot Avg. Primary Fresh wt. of Dry wt. of No. No. of length (cm.) Root length plantlet plantlet Leaves (cm.) (gm.) (gm.) 1 CMS-A (0246) CMS-B P4-Male (2+3) LCA *(2+3)- F1 Hybrid B] Second tissue culture experiment for biomass production: 1. Number of days required for seed germination: No seed germination was observed till 16 days from the date of inoculation of seeds. After 16 days different genotypes started showing germination at different rates and on different days. The first genotype which was observed to be germinated on 17 th day from the date of inoculation was the 301-F GMS genotype. It was followed by germination of second GMS genotype 0215-F on 18 th day. GMS-based genotypes like 301-F, 0215-F germinated later on after CMS-based genotypes but after germination very fast and vigorous growth pattern was seen in their case. 2. Average shoot length: The maximum average shoot length was shown as 4.8cm and 4.2cm in both genetic male sterile genotypes (301-F and 0215-F) and 5.2cm in cytoplasmic male sterile genotype (302-A) respectively. Less average shoot length was shown as 4.0cm in hybrid-301-f*(301-male) genotype (Table 3). 3. Average number of leaves: The maximum average number of leaves was shown 7 and 8 in both genetic male sterile genotypes (301-F and 0215-F) respectively and 7 in cytoplasmic male sterile genotype (302-A). Average number of leaves in maintainer genotype (302-B) was observed as 6. Average number of leaves observed was very less in F1 hybrid-301- F*(301-Male) and the number was observed as 3 (Table 3). 4. Average root length (including only the primary roots length): The maximum average root length was shown as 12.5cm and 9.0cm in both genetic male sterile genotypes (301-F and 0215-F) respectively and it was observed as 7.8cm in cytoplasmic male sterile genotype (302-A). Average root length in maintainer genotype (302-B) was observed as 6.9cm. Average root length was observed as 2.7cm and 2.6 in case of 301-male and 301-F*(301- Male)-F1 hybrid respectively (Table 3). 5. Average fresh weight of plantlet: The maximum average fresh weight is shown as 0.19gm and 0.12gm in both genetic male sterile genotypes (301-F and 0215-F), wherein it is 0.14gm and 0.13gm in cytoplasmic male sterile genotype (302-A) and its CMS-based maintainer genotype (302-B) respectively (Table 3). 6. Average dry weight of plantlet : The maximum average dry weight was shown as 0.008gm and 0.10gm in both two genetic male sterile genotypes (301-F and 0215-F) respectively. Average dry weight was observed as 0.007gm and 0.008gm in cytoplasmic male sterile genotype (302-A) and its CMS-based maintainer genotype (302-B) respectively. Average dry weight for both 301-male and 301-F*(301-Male) F1-hybrid was observed as 0.003gm (Table 3). Pg. 152

5 Table 3: Observations of different genotypes including genetic male sterile genotypes, cytoplasmic male sterile genotype, restorer genotype, GMS-based hybrid, CMS-maintainer genotype, and fertile genotype after conducting second tissue culture experiment. Sr. No. Genotype Avg. No. of Leaves Avg. Shoot length (cm.) Avg. Primary Root length (cm.) Fresh wt. of plantlet (gm.) Dry wt. of plantlet (g) F F A Male F*(301- Male)-F Hybrid B LCA In general sense, the term biomass is used by scientists to describe the amount or mass of organic matter in a prescribed area at a given point in time. This measure of biological matter includes material formed above and below ground. Sometimes, scientists eliminate the moisture factor by measuring the weight of the harvested product on a dry matter basis after drying the product in an oven prior to being weighed. Biological yield, thus may be defined as the total dry matter produced per plant or per unit area (i.e., biomass). Researchers use this measurement of yield in agronomic, physiological, and plant breeding research to indicate dry matter accumulation by plants. All yields are at first biological yield. To increase yield, the breeder may breed for increased biomass and efficient partitioning of assimilates [8]. Considering the use of male sterile genotypes in plant breeding research, biomass production shows different effect on the yield or productivity of the plant. Generally, genotypes which are cytoplasmic male sterile genotypes (CMS), and genetic male sterile genotypes (GMS) show vigorous vegetative growth as compared to fertility restorer/fertile genotypes or CMS/GMS based F1-hybrid genotypes. Male sterile genotypes grow vegetatively until they get pollen deposition from external sources since they are incapable of producing viable pollen themselves. On the contrary, normal fertile genotypes, restorer genotypes and F1-hybrid genotypes are fertile themselves and get fruit setting early at maturity by restricting themselves from vigorous vegetative growth, thereby such normal male fertile genotypes show less vegetative growth compared to male sterile genotypes. This vigorous biomass production by male sterile genotypes is a superior economic character in plants where vegetative part is of economic significance, example, lettuce, cabbage, spinach, etc. but, it can be a detrimental character for plants where plant product is a fruit as in Capsicum, tomato, etc. which ultimately affect the final fruit setting and fruit production in terms of yield of the plant. Thus in this paper, we are presenting our observations in Capsicum annum L. plant regarding in vitro biomass production character of male sterile genotypes (CMS and GMS), fertility restorer/fertile genotypes along with their F1- hybrid genotypes. The conducted study establishes the fact that in vitro biomass production is more in male sterile genotypes (GMS and CMS) and CMS based maintainer genotypes of Capsicum annum L. as compared to fertile genotypes. From our study, we observed that the in vitro vegetative growth or biomass in cytoplasmic male sterile genotype (CMS-A (0246)), and its maintainer genotype (CMS-B) was found to be more as compared to restorer genotype or normal fertile genotypes [(P4-Male (2+3) or LCA-353]. CMS-based F1-hybrid [0246*(2+3)] showed very less biomass as fruit setting in fertile plants takes place early at maturity without leading to vigorous vegetative growth/biomass (Table 2). Pg. 153

6 Likewise vegetative growth or biomass is more in cytoplasmic male sterile genotype (CMS-302-A) and genetic male sterile genotypes (GMS-301-F and 0215-F) which was found to be more as compared to GMS based restorer genotype (301- Male) or normal fertile genotype (LCA-353), but GMS based F1 hybrid [(301-F*(301-Male)] was found to produce less biomass just like fertility restorer genotype (301-Male) which indicates that fertile plants themselves show fruit setting early at maturity without leading to vigorous vegetative growth/biomass. On the contrary the maintainer genotype (302-B) shows significantly more biomass as seen in (301-F) since they are genotypes which maintain sterility in CMS genotype (Table 3). CONCLUSION: From the presented study, we recommend to use in vitro vegetative growth rate or biomass production parameter by adopting tissue culture techniques in case of Capsicum annum L. before conducting breeding programs involving male sterile genotypes (CMS or GMS) for production of higher fruit yielding Capsicum annum L. varieties, wherein such tissue culture technique is simple, less time consuming and less expensive. Such consideration may help breeders to decide upon choosing of superior male sterile genotypes (CMS or GMS) and an appropriate male parent genotype for getting final higher fruit setting, fruit production in CMS/GMS based F1-hybrid. REFERENCES: 1. Kumar S, Rai M. Chile in India. Chile Pepper Institute Newsletter, 2005; 22: Ravishankar GA, Suresh B, Giridhar P, Rao SR, Johnson TS (2003). Biotechnological studies on capsicum for metabolite production and plant improvement. In: DE, Amit Krishna ed. Capsicum: The genus Capsicum. Harwood Academic Publishers, UK, pp Zhao Hualun, Ding Liping, Sun Jiebo & Qian Zhilong (1995). Selection of Capsicum male sterility lines 21A, 8A AND 17A and their authentication. Jiangsu Agricultural Sciences, 1995 (1): Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1962; 15: Madhuri V, Rajam MV. Apical shoot meristemculture in red pepper (Capsicum annuum L.). J Plant Biochem Biotech. 1993; 2: Christopher T, Rajam MV. In vitro clonal propagation of Capsicum spp. Plant Cell Tissue Organ Cult. 1994; 38: Mahmood M, Atan Z, Kow CW. Studies on tissue culture of different chilli cultivars (Capsicum annuum L.). Asia-Pacific J Mol Biol Biotech. 1995; 3: George A. Principles of Plant Genetics and Breeding Pg. 154

7 How to cite this article APA style Suryawanshi, M.K., Baig, M.M.V. (2013). Biomass production in male sterile and fertile genotypes of capsicum annum l. International Journal of Pharma Bioscience and Technology, 1(3), Elsevier Harvard style Suryawanshi, M.K., Baig, M.M.V., Biomass production in male sterile and fertile genotypes of capsicum annum l. Int. J. Pharm. Biosci. Technol. 1, Vancouver Style Suryawanshi MK, Baig MMV. Biomass production in male sterile and fertile genotypes of capsicum annum l. Int J Pharm Biosci Technol. 2013;1(3): To receive bibliographic information in RIS format (For Reference Manager, ProCite, EndNote): Send request to: ijpbst@yahoo.com To return to the first Page: Click Here Pg. 155