USE OF MALE STERILITY AND SYNTHESIS OF MAINTAINER LINE FOR HYBRID SEED PRODUCTION IN ONION ( ALLIUM CEPA L . )

A field experiment was carried out and one cytoplasmic genetic male sterile line (Smsms) and two fertile lines were identified as maintainer lines (Nmsms). These two crossed materials namely 004 (Shallot x Red creole) and 008 (Shallot x Red pinoy) produced 100 per cent male sterile progeny in full sib and backcross generations. The Shallot x Taherpuri/Suksagor and Shallot x Hazera-202/Hazera-203 cultivar produced both male fertile and male sterile segregating progenies. It indicated that these materials are probably determined by dominant and recessive independently acting genes which was resulting the genetically impure lines. All other crossed materials produced 100 per cent male fertile progeny upon crossing with shallot. Thus, the materials Red creole and Red pinoy can be used as maintainer line for “Shallot”. The performance of 904 F1 and 905 F1 hybrids over check (Taherpuri) and better parent was found to be preferably better using these CMS system.


INTRODUCTION
Onion (Allium cepa L.) is an important spice around the world and grown as a winter crop in Bangladesh.Average yield 3.45 t/ha in Bangladesh and 45 t/ha in developed country (FAO 2002).Low yielding variety and Inadequate supply of quality seed is the main constraint to its higher production.Due to lack of high yielding variety, Bangladesh importing onion bulb worth of about BDT 400-500 crores in every year (Prothom Alo, July 11, 2004).It is highly cross pollinated crop and sensitive to day length and temperature (Pike 1986).Early stage for vegetative growth needs cooler weather and formation of bulb is enhanced by high temperature.Hybrid varieties play a vital role in increasing vegetable production due to their high yield potential, early maturing, superior quality, disease and pest resistance attributes (Nagaraju et al. 2017).MS mechanism is one of the tools for hybrid variety development and it is the most significant mechanism for improving the yield and higher returns of onion (Pike, 1986, Kalloo, 1988).First CMS plant was reported within the progenies of an onion cultivar Italian Red (Jones and Emsweller, 1936) and male sterility was under the control of single recessive nuclear restorer locus (Jones and Clarke, 1943).A good male sterile line can be obtained by repeated backcrossing, where local maintainer is very essential (Chatterjee, 1986).Red color onion bulb is not preferred by the Bangladeshi farmers and consumers.So, this offers a great scope for improving the production of this crop in Bangladesh through CMS breeding.In this consequence however, the present study was conducted for searching the maintainer line from distance heterotic pole for the development of hybrid onion variety which could be performed as better yielder than our local cultivar and to fight against different agro-climatic condition.

Materials and Methods
Bulbs of "shallot" (Fig. 2) and its probable maintainer (Nmsms) were collected from the open market of Philippines, Bangladesh and Thailand in November 2006.Shallot bulbs were vernalized at 10 o C for four weeks as this cultivar do not flower or poorly flower under Bangladesh conditions.Total eleven male fertile both local and exotic cultivars viz.Taherpuri, Suksagor, Batanes, Red pinoy, NZ-2, Red creole, Hazera-202, Hazera-203, Nueventa, Light red and Nasik red were grown at the same time.At flowering stage the "Shallot" was crossed with fertile pollen during the winter season of 2007-2008 at Research and Development wing (R&D) of Lal Teer Seed Ltd., Gazipur.In 2009-2010 at flowering stage both Male Sterile (MS) and Male Fertile (MF) plants were identified through the study of flower morphology and pollen viability test.Performances of MS plants from F1 families in respect of days to bolting and days to flowering were recorded for synchronization.In addition few F1 plants of 004 (Shallot x Pinoy) and 008 (Shallot x Red Creole) were back crossed with their recurrent and expected pollen parents.Total six crosses as F1 were evaluated during 2012-2013 in respect of yield performance against check variety (Taherpuri).Total methodologies were cited as below: 1 st year: Bulb to Seed  Growing the selected bulbs of expected MS lines and expected B -lines in alternate rows  Selection of male sterile plants based on pollen sterility using 1% acetocarmin dye. Crossing between MS (A-line) and expected B-lines.
2 nd year: Seed to bulb  Growing the bulbs of the cross between A-line and expected B-line  Selection of bulbs based on desirable characters like color  Selection of bulbs including pungency and storability 3 rd year: Bulb to Seed  Planting of selected bulbs based on storage quality and pungency  Identifying MS plants which will be repeatedly backcrossed with B-line (maintainer line)  Crossing between A-line with expected C-line (Restorer line).
4rth year: Seed to bulb  Growing and selecting bulbs based on desirable characters. Storing the selected bulbs in ambient conditions.5 th year: Bulb to seed  The progeny from A*C will show male sterility or fertility with desirable heterosis

RESULTS AND DISCUSSION
The plants of all the crossed families flowered.The percentage of male sterile (MS) plants (Fig. 3) in the families resulting from the crosses between shallot and male fertile (MF) plants varied from 0 to 100 (Table 1.).Two exotic cultivars (Red Pinoy and Red creole) were identified as maintainer lines as these cultivars were found to posses' recessive chromosomal gene (msms) responsible for male sterility.These results revealed that the pollen parents which possessed msms genes could be used as a maintainer line of "Shallot".The exotic cultivars viz.Batanes, NZ-2, Nueventa, Light red and Nasik red were found to posses the dominant gene (MsMs) responsible for restoration of male fertility in the sterile plants.On the other hand exotic cultivar Hazera-202 and 203 as well as our local cultivar Taherpuri and Suksagor were found both male fertile and male sterile segregating progenies in a certain extent which indicated that these materials were probably determined by dominant and recessive independently acting genes resulting the genetically impurity or the effect of temperature.The materials Hazera-202 and Hazera-203 produced 50 percent male fertile and 50 percent male sterile progenies upon crossing with shallot plants.Furthermore, our local cultivar Taherpuri exhibited 90 percent male fertile and 10 percent male sterile along with Suksagor which was in 75:25 fertility sterility ratios indicating the presence of Msms genes in their cytoplasm.
The performance of 904 F1 and 905 F1 hybrids (Table 2 and Fig. 4) over check and better parent was found to be preferably better using CMS system.The local pollen parent has the potentiality to maintain the sterility of shallot.The flowering period of shallot and local lines are naturally well synchronized.Almost similar findings were also reported by Mohsin (1996) and Hossain (1995) who used the "Ogura radish" which is male sterile lines.For the production of hybrid, A, B and C lines will be required (Fig. 1 & 5).Line A is with shallot (Smsms), Line B is the Red Creole and Red Pinoy (Nmsms) and line C is the (Taherpuri).

Genetic constituents of CMS and maintainer lines CMS line or A-line: S msms (S is sterile)
Maintainer or B-line: N msms (N is fertile) Genetic constituents of A and B line is identical except the fertility which is govern by a single gene presence in B line.By the way of illustration A-line is genetically male sterile with recessive gene consequently B-line is genetically male fertile with recessive gene

How to develop a maintainer line, if not available in the nature
 1 st : Smsms * NMsMs………….>SMsms(all male fertile)  2 nd : NMsMs * SMsms…………..> NMsMs, NMsms (all male fertile)  3 rd : First Progeny test: So, Nmsms is identified as maintainer which is able to maintain the CMS mechanism.CMS line and new B line (Nmsms) to be planted in isolation for repeated backcrossing to transfer all characters of B line except fertility.

Development of A-line
 Backcross breeding method is applied for development of A-line using identified Bline and CMS line. By the way B-line should be selfed for its maintaining  After 5-6 backcross CMS line will be converted as expected A-line. 1 st yr: 50%, 2 nd yr: 75%, 3 rd yr: 87.5%,4 th yr: 93.75, 5 th yr: 96.875, 6 th yr: 98.4375, 7 th yr: 99.21875  A and B line is isogenic.Difference is only male fertility and sterility.The characteristics of the "local lines" are to be transferred in the "shallot" by backcrossing.For this, another three to four backcrosses to "local lines" will be required.Certain precautions such as frequent insect pollination should be allowed and rouging of male fertile plants from CMS line should be done timely to avoid unexpected out crossing.So, considering the genetics of the male sterility mechanism, further studies should be taken towards synthesis of maintainer line from local cultivar as well as hybrid variety development for better stability along with better yield and to combat climatic fluctuation in Bangladesh.

3. 1 :
Fig 1.Schematic diagram of hybrid variety of onion development using CMS mechanism.
of A, B and C/R lines  Components of three lines for hybrid development: 1. A-line (Male sterile line or CMS line) 2. B-line (Male fertile line) 3. C/R-line (Male fertile) Genetic makeup of components lines: A-line: This CMS is genetically pure and male sterile.It possesses nuclear recessive gene and sterile cytoplasm (Smsms) B-line: Identified B-line is fertile and possesses nuclear recessive gene with fertile cytoplasm (Nmsms) Before backcross A-line and B-line is morphologically dissimilar

Fig
Fig 2. Sprouted bulb of shallot Onion Fig 3. Male sterile flowers (left) and male fertile (right) flowers in umbel of onion