Method for obtaining lettuce haploid plant through tissue culture
A tissue culture and plant technology, applied in the field of biological breeding, can solve the problems of easy browning of callus, no relevant reports, few successful cases, etc., achieve new variety selection and genetic breeding promotion, low technical difficulty, and simple steps Effect
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Embodiment 1
[0027] Embodiment 1, comparison after inoculation of lettuce flower buds of different specifications
[0028] Harvest bolted lettuce flower branches, put them in a refrigerator at 4°C for 48 hours as a pretreatment, and then cut them into smaller branches and place them on the ultra-clean workbench (see figure 1 ); It can be seen that lettuce flower buds generally have three specifications, smaller young flower buds with a diameter of 2mm or less, lettuce flower buds with a diameter of about 3mm, and lettuce flower buds that are about to open above 4mm, and are sterilized with 10% sodium hypochlorite solution added Tween 20 15min; finally wash 4-5 times with sterile water, dry the surface water with sterile filter paper, use tweezers to poke the sepals and peel off the flowers ( figure 2 Cauliflower extracted by tweezers), inoculated on MS medium containing 1mg / L 6-BA and 2mg / L NAA for callus induction. Follow-up observations found that more calluses were formed in young flo...
Embodiment 2
[0029] Embodiment 2, subculture and differentiation culture of lettuce callus
[0030] After the cauliflower of suitable size inoculated in Example 1 was cultivated in the induction medium (MS medium containing 6-BA1mg / L+NAA2mg / L) for about 14d, it could be seen that there was obvious callus growth, and after about 25d, it was transferred to The callus was subcultured and differentiated in MS medium containing 2mg / L 6-BA and 0.1mg / LNAA. As a contrast: the other part continues to be inoculated in the original formula medium, and in the follow-up cultivation, it is found that its growth is prone to browning, and it is difficult to grow clustered buds, and the callus is more likely to vitrify (see Figure 4 Browning lettuce callus on the left). It did not perform as well as subdifferentiation medium with increased 6-BA and reduced NAA concentrations ( Figure 4 The middle growth situation is better), so the MS medium of 2mg / L 6-BA and 0.1mg / LNAA is finally selected as the subcu...
Embodiment 3
[0031] Embodiment 3, lettuce differentiation emergence and ploidy identification
[0032] Embodiment 2 After subculture and differentiation culture, the lettuce callus grows clusters of shoots (see Figure 4 Clustered shoots grew in the far right callus), the clustered shoots were separated and inoculated into rooting medium (MS medium containing NAA 0.05 mg / L) to practice seedling rooting, and the leaves were taken and chopped and then doubled by flow cytometry. Sex detection, and finally haploid lettuce was obtained.
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